JP5292304B2 - Peptide nanoparticles and uses thereof - Google Patents

Abstract

The present invention provides nanoparticle compositions including one or more peptides. The present invention achieves transdermal delivery of such peptides without the need for peptide modification, or for use of chemical or mechanical abrasion or disruption of skin.

Description

RELATED APPLICATIONS This application is directed to US Provisional Patent Application No. 60 / 872,206 ("the '206 application"), filed December 1, 2006, under US Patent Act §119 (e), Claim priority. The entire contents of this “'206 Application” are incorporated herein by reference.

BACKGROUND OF THE INVENTION Peptides have been shown to have beneficial cosmetic and therapeutic effects on the skin. Experimental models have shown that short peptides (up to 30 amino acids long) stimulate collagen growth in the extracellular matrix of the skin, which improves the appearance of the skin and also damages the damaged skin. Healing can be further improved (Non-Patent Document 1; this is incorporated herein by reference). Modified peptides have also been shown to make wrinkles less noticeable by regulating enzymes that affect muscle contraction in the muscles under the skin that contribute to wrinkle formation (2). Is incorporated herein by reference).

  However, the main problem in embodying the possible cosmetic and therapeutic effects of these peptides in humans is that they are biologically active across the outer skin barrier (stratum corneum), such as at the extracellular matrix or below Delivering peptides transcutaneously into certain muscles (Non-Patent Document 3; which is incorporated herein by reference). In order to achieve transdermal delivery of peptides in humans, peptides must be chemically modified by adding chemical moieties such as, but not limited to, acetyl and / or palmitoyl groups (Robinson). Et al., Above). Such chemical modifications are disadvantageous because they are expensive and time consuming, which adversely affects the commercial production of products containing these peptides. Chemical modification of a peptide reduces its biological activity by reducing its ability to bind at a cellular receptor site of biological activity (eg, by steric hindrance), thereby reducing its effectiveness. There is also a fear. Biologically ineffective peptides are also less effective for cosmetic or therapeutic purposes. Biologically ineffective peptides require relatively high levels of administration to achieve the desired biological effect (as it may have been), which is the commercial production of the product This is a cost disadvantage.

Katayama et al., 1993, J. MoI. Biol. Chem. 268: 9941 Lupo, 2005, Dermatol. Surg. 31: 832 Robinson et al., 2005, International J. MoI. Cosmetic Science 27: 155

SUMMARY OF THE INVENTION The present invention incorporates unmodified short peptides (2-30 amino acids long) that are biologically active substances in skin (including epidermis and dermis), subcutaneous tissue (including adipose tissue) and adjacent muscle. The nanoparticles will be described.

  The nanoparticles of the present invention can be applied to the subject's skin. In some embodiments, the nanoparticles of the present invention provide for transdermal delivery of incorporated peptides to a subject.

  The nanoparticles of the present invention can be applied to the skin as a simple suspension or dispersion, or mixed with one or more excipients, including but not limited to emollients, nutritional lotions. , Cleansing lotions, cleansing creams, emulsions, emollient lotions, massage creams, emollient creams, makeup bases, lipsticks, facial packs or gels, detergent formulations (eg shampoos, rinses, body cleaners, hair tonics and soaps) and It can be prepared as a formulation for skin compositions (eg lotions, ointments, gels, creams, patches and sprays).

Thus, the present invention provides systems and compositions for delivering unmodified peptides transdermally. Among the many advantages of the present invention is the ability to deliver peptides without the need for injection and without the need for mechanical or chemical wear or alteration of the skin. Another advantage is that unmodified peptides can be utilized, thereby simplifying the production of the cosmetic and / or pharmaceutical products of the present invention, reducing its cost, and preserving the biological activity of the peptides. It is included.
For example, the present invention provides the following items.
(Item 1)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, wherein the nanoparticles are biological in the skin, subcutaneous tissue or adjacent muscle Suspension or dispersion comprising at least one unmodified peptide of 2 to 30 amino acids in length having a pharmacological activity.
(Item 2)
The suspension or dispersion of item 1, wherein a majority of the particles have a diameter in the range of about 10 to about 250 nm.
(Item 3)
The suspension or dispersion of item 1, wherein a majority of the particles have a diameter in the range of about 10 to about 200 nm.
(Item 4)
The suspension or dispersion of item 1, wherein the majority of the particles have a diameter in the range of about 10 to about 150 nm.
(Item 5)
The suspension or dispersion of item 1, wherein a majority of the particles have a diameter in the range of about 10 to about 120 nm.
(Item 6)
The suspension or dispersion of item 1, wherein a majority of the particles have a diameter in the range of about 10 to about 100 nm.
(Item 7)
The suspension or dispersion of item 1, wherein the majority of the particles have a diameter in the range of about 10 to about 50 nm.
(Item 8)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence comprising KTTKS Suspension or dispersion.
(Item 9)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of from about 10 to about 300 nm, wherein the nanoparticles comprise an unmodified peptide whose amino acid sequence comprises EYKTTKSSRL Suspension or dispersion.
(Item 10)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of from about 10 to about 300 nm, wherein the nanoparticles comprise the amino acid sequence VIEYKTTK Suspension or dispersion.
(Item 11)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence comprising KTTK Suspension or dispersion.
(Item 12)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence comprising GKTVIEYKTTTKS Suspension or dispersion.
(Item 13)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence GKTVIEYKTTTSSRL Suspension or dispersion.
(Item 14)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, wherein the nanoparticles comprise an amino acid sequence WGKTVIEYKTTKSSRLPIID Suspension or dispersion.
(Item 15)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence CTSHTGAWGKTVEYKTTTKS Suspension or dispersion.
(Item 16)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence comprising TTKS Suspension or dispersion.
(Item 17)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an EEMQRR amino acid sequence Suspension or dispersion.
(Item 18)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising a non-amino acid sequence comprising a gastrin releasing peptide. A suspension or dispersion containing the modified peptide.
(Item 19)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence comprising VGVAPG Suspension or dispersion.
(Item 20)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an YYRADA amino acid sequence Suspension or dispersion.
(Item 21)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an unmodified peptide whose amino acid sequence comprises GHK Suspension or dispersion.
(Item 22)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence comprising TTKS Suspension or dispersion.
(Item 23)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an interferon in its amino acid sequence Suspension or dispersion.
(Item 24)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, wherein the nanoparticles have a non-amino acid sequence comprising an interferon-inducing substance. A suspension or dispersion containing the modified peptide.
(Item 25)
A suspension or dispersion of nanoparticles comprising a population of particles, the majority of the particles having a diameter of about 10 to about 300 nm, the nanoparticles comprising an amino acid sequence P144 Suspension or dispersion.
(Item 26)
The suspension or dispersion according to any one of Items 1 to 18, wherein the nanoparticles are nanoemulsions.
(Item 27)
27. A suspension or dispersion according to item 26, wherein the nanoemulsion comprises at least one oil, at least one surfactant, water and an unmodified peptide.
(Item 28)
27. A suspension or dispersion according to item 26, wherein the nanoemulsion comprises a single oil, a single surfactant, water and an unmodified peptide.
(Item 29)
27. A suspension or dispersion according to item 26, wherein the nanoemulsion comprises a single oil, a single surfactant, water and an unmodified peptide.
(Item 30)
The oil is almond, apricot, avocado, babas, bergamot, black currant seed, borage, cadet, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed , Emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, loofah, grape seeds, hazelnuts, hyssop, jojoba, kukui seeds, lavandin, lavender, lemon, lychee aqaba, macadamia nuts, aoi, mango seeds, meadowweed seeds , Mink, nutmeg, olive, orange, orange luffy, palm, palm kernel, peach seed, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasanqua, savory, sea buckthorn, Maize, Shea Butter, Silicone, Soybean, Sunflower, Tea Tree, Thistle, Camellia, Vetiver, Walnut and Wheat Germ Oil; Butyl Stearate; Caprylic Acid Triglyceride; Capric Acid Triglyceride; Cyclomethicone; Diethyl Sebacate; Dimethicone 360; Item 27- selected from the group consisting of isopropyl myristate; mineral oil; octyldodecanol; oleyl alcohol; silicone oil; short chain triglyceride; medium chain triglyceride; Labrafac WL1349; long chain triglyceride; 30. The suspension or dispersion according to any one of 29.
(Item 31)
30. The suspension or dispersion according to any one of items 27 to 29, wherein the oil is soybean oil.
(Item 32)
30. The suspension or dispersion according to any one of items 27 to 29, wherein the oil is a medium chain triglyceride.
(Item 33)
30. A suspension or dispersion according to any one of items 27 to 29, wherein the oil is Labrafac WL1349.
(Item 34)
The surfactant is phosphoglyceride; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; Diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surface active fatty acids such as palmitic acid or oleic acid; fatty acids; fatty acids Monoglycerides; fatty acid dinoglycerides; fatty acid amides; sorbitan trioleate (Span 85) Polysorbate; sorbitan monolaurate (Span20); polysorbate 20 (Tween-20); polysorbate 60 (Tween-60); polysorbate 65 (Tween-65); polysorbate 80 (Tween-80); polysorbate 85 (Tween-85); Surfactin; poloxamer; sorbitan fatty acid esters such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; Dipalmitoyl phosphatidylglycerol; stearylamine; dodecylamine; Oxadecyl-amine; Acetyl palmitate; Glycerol ricinoleate; Hexadecyl stearate; Isopropyl myristate; Tyloxapol; Poly (ethylene glycol) 5000-phosphatidylethanolamine; Poly (ethylene glycol) 400-monostearate; Phospholipids; High 30. Suspension according to any one of items 27 to 29 selected from the group consisting of synthetic and / or natural detergents having surfactant properties; deoxycholates; cyclodextrins; chaotropic salts; and ion-pairing agents Dispersion.
(Item 35)
30. The suspension or dispersion according to any one of items 27 to 29, wherein the surfactant is Tween.
(Item 36)
36. A suspension or dispersion according to any one of items 27 to 35, wherein the weight ratio of surfactant to oil ranges from 0.5: 1 to about 2: 1.
(Item 37)
36. A suspension or dispersion according to any one of items 27 to 35, wherein the weight ratio of surfactant to oil is about 0.25: 1.
(Item 38)
36. The suspension or dispersion according to any one of items 27 to 35, wherein the weight ratio of surfactant to oil is about 0: 5: 1.
(Item 39)
36. A suspension or dispersion according to any one of items 27 to 35, wherein the weight ratio of surfactant to oil is about 1: 1.
(Item 40)
36. A suspension or dispersion according to any one of items 27 to 35, wherein the weight ratio of surfactant to oil is about 2: 1.
(Item 41)
36. A suspension or dispersion according to any one of items 27 to 35, wherein the weight ratio of surfactant to oil is about 3: 1.
(Item 42)
The suspension or dispersion according to any one of Items 1 to 18, wherein the nanoparticles are nano micelles.
(Item 43)
Item 51. The item according to any one of items 1 to 42, further comprising at least one additional ingredient that, together with the suspension or dispersion, produces a cream, oil, ointment, gel, spray, lipstick or sunscreen. Suspension or dispersion.
(Item 44)
Providing a subject;
Administering to the subject's skin a suspension or dispersion according to any one of items 1 to 43 in an amount such that cosmetic or therapeutic utility is obtained;
Including methods.
(Item 45)
Providing a subject;
Administering a suspension or dispersion according to any one of items 1 to 43 to the subject's skin in an amount effective to thicken the subject's skin or subcutaneous tissue;
Including methods.
(Item 46)
Providing a subject suffering from, susceptible to or exhibiting at least one symptom of fine skin lines;
Effective for treating, reducing, improving, mitigating, delaying its development, preventing its progression, reducing its severity, or reducing its incidence, said fine skin lines Administering the suspension or dispersion according to any one of the items 1 to 43 to the skin of the subject;
Including methods.
(Item 47)
Providing a subject suffering from, susceptible to or exhibiting at least one symptom of facial wrinkles;
Effective amount 1 to treat, reduce, improve, alleviate facial wrinkles, delay its development, prevent its progression, reduce its severity, reduce its incidence -Administering the suspension or dispersion according to any one of -43 to the skin of the subject;
Including methods.
(Item 48)
Providing a subject;
Administering a suspension or dispersion according to any one of items 1 to 43 to the subject's skin in an amount effective to improve the appearance of the subject's skin;
Including methods.
(Item 49)
Providing a subject;
Administering a suspension or dispersion according to any one of items 1-43 to the subject's skin in an amount effective to maintain the appearance of the subject's skin;
Including methods.
(Item 50)
Providing a subject suffering from trauma;
44. Suspension according to any one of items 1 to 43, in an amount effective to treat, reduce, ameliorate, alleviate, inhibit the progression of, or reduce the severity of said trauma. Or administering the dispersion to the subject's skin;
Including methods.
(Item 51)
Provide a subject suffering from, susceptible to or exhibiting at least one symptom of hypertrophic scars, keloids, localized sclerosis, systemic sclerosis or other conditions characterized by excessive accumulation of extracellular matrix Steps,
Treat, reduce, ameliorate, alleviate at least one symptom associated with hypertrophic scars, keloids, localized sclerosis, systemic sclerosis, or other conditions characterized by excessive accumulation of extracellular matrix, 44. Suspension or dispersion according to any one of items 1-43 in an amount effective to delay its occurrence, prevent its progression, reduce its severity, or reduce its incidence Administering the liquid to the subject's skin;
Including methods.
(Item 52)
52. The method according to any one of items 44 to 51, wherein the administering step is performed manually.
(Item 53)
52. A method according to any one of items 44 to 51, wherein the administering step is performed with a gloved finger.
(Item 54)
52. The method according to any one of items 44 to 51, wherein the administration step is performed by an unprotected hand.
(Item 55)
52. A method according to any one of items 44 to 51, wherein the administering step is performed on a device.
(Item 56)
52. A method according to any one of items 44 to 51, wherein the administering step is performed with an adhesive patch.
(Item 57)
52. The method according to any one of items 44 to 51, wherein the administering step is performed with a spatula.
(Item 58)
52. The method of any one of items 44 to 51, wherein the administering step is performed with a swab.
(Item 59)
52. A method according to any one of items 44 to 51, wherein the administering step is performed with a needleless syringe.
(Item 60)
52. Any one of items 44 to 51, wherein the administering step is performed using a device that allows the suspension or dispersion to be applied to a target site on the skin without applying to the non-target site of the skin. The method described in 1.
(Item 61)
61. The method of any one of items 44-60, wherein all of the unmodified peptide penetrates the skin.
(Item 62)
61. The method of any one of items 44-60, wherein at least 99% of the unmodified peptide penetrates the skin.
(Item 63)
61. The method of any one of items 44-60, wherein at least 95% of the unmodified peptide penetrates the skin.
(Item 64)
61. The method of any one of items 44-60, wherein at least 90% of the unmodified peptide penetrates the skin.
(Item 65)
61. The method of any one of items 44-60, wherein at least 75% of the unmodified peptide penetrates the skin.
(Item 66)
61. The method of any one of items 44-60, wherein at least 50% of the unmodified peptide penetrates the skin.
(Item 67)
61. The method of any one of items 44-60, wherein at least 25% of the unmodified peptide penetrates the skin.
(Item 68)
61. The method of any one of items 44-60, wherein at least 10% of the unmodified peptide penetrates the skin.
(Item 69)
61. The method of any one of items 44-60, wherein at least 1% of the unmodified peptide penetrates the skin.
(Item 70)
Providing a premix comprising oil, surfactant, water and unmodified peptide;
Subjecting the premix to high shear or high pressure homogenization for a period and under conditions that result in a suspension or dispersion according to any one of items 1-43;
Including methods.
(Item 71)
71. The method of item 70, wherein subjecting the premix to high shear or high pressure homogenization is performed by microfluidization.
(Item 72)
71. The method of item 70, wherein the weight ratio of oil to surfactant in the premix ranges from about 0.5: 1 to about 2: 1.
(Item 73)
71. The method of item 70, wherein the weight ratio of oil to surfactant in the premix is about 0.25: 1.
(Item 74)
71. The method of item 70, wherein the weight ratio of oil to surfactant in the premix is about 0: 5: 1.
(Item 75)
71. The method of item 70, wherein the weight ratio of oil to surfactant in the premix is about 1: 1.
(Item 76)
71. The method of item 70, wherein the weight ratio of oil to surfactant in the premix is about 2: 1.
(Item 77)
71. The method of item 70, wherein the weight ratio of oil to surfactant in the premix is about 3: 1.
(Item 78)
At least one skin sample; and
At least one suspension or dispersion comprising at least one unmodified peptide
Providing steps, and
Administering the suspension or dispersion to a skin sample;
Monitoring the effect of the suspension or dispersion on the appearance of the skin sample relative to the effect of the control substance;
A method of identifying an unmodified peptide that improves the appearance of skin, comprising:
(Item 79)
79. The method of item 78, wherein the control substance is a substance that improves the appearance of a skin sample.
(Item 80)
79. The method of item 78, wherein the control substance is a substance that does not improve the appearance of the skin sample.

It is a histological analysis of mice treated with peptide nanoparticles. It is a microscope picture which shows the skin tissue dye | stained by Masson's trichrome dyeing | staining. The average histological score was 2.33 out of 4 possible in the control group (nanoparticle formulation without pentapeptide). The average histological score was 3.67 out of 4 possible in the treatment group (nanoparticle formulation with pentapeptide).

Definitions Abrasion: As used herein, the term “wear” refers to any means that alters, disrupts, removes or destroys the top layer of skin. In some embodiments, abrasion refers to a mechanical means that modifies, collapses, removes or destroys the top layer of skin. In some embodiments, abrasion refers to a chemical means that modifies, disintegrates, removes or destroys the top layer of skin. To name a few, agents such as exfoliants, particulates (eg, magnesium or aluminum particles), acids (eg, α-hydroxy acids or β-hydroxy acids), alcohols can cause wear. . In general, for example, Donovan (eg, US Patent Publication Nos. 2004/009180 and 2005/175636 and PCT Patent Publication WO 04/06954, all of which are incorporated herein by reference) and Graham (eg, US Pat. No. 6,939). , 852 and U.S. Patent Publication No. 2006/093624; both of which are incorporated herein by reference) and the like are believed to cause wear. Of course, one of ordinary skill in the art will be able to cause wear when a particular agent is present at one concentration or with one or more other agents, but may cause wear under different circumstances. It will be understood that cannot. Thus, whether a particular material is a “wear agent” will depend on the situation. One skilled in the art can readily assess wear. For example, it can be assessed by observation of skin redness or inflammation and / or histological examination of the skin showing alteration, collapse, removal or erosion of the stratum corneum.

Amino acid: As used herein, the term “amino acid”, in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, the amino acid has the general structure H ₂ N—C (H) (R) —COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid. In some embodiments, the amino acid is a D-amino acid. In some embodiments, the amino acid is an L-amino acid. “Standard amino acid” refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. Amino acids, including carboxy-terminal and / or amino-terminal amino acids in the peptide, may be methylated, amidated, acetylated, and / or other chemistries that can alter the circulating half-life of the peptide without adversely affecting its activity. It can be modified by replacement with a group. However, as described herein, the present invention is specifically directed to “unmodified peptides” which refer to peptides that have not undergone chemical modification to facilitate or achieve transdermal delivery. Amino acids can participate in disulfide bonds. The term “amino acid” is used interchangeably with “amino acid residue” and can refer to a free amino acid and / or an amino acid residue of a peptide. Whether it refers to a free amino acid or a residue of a peptide will be apparent from the context in which the term is used.

  Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human at any stage of development. In some embodiments, “animal” refers to any developmental animal other than humans. In certain embodiments, the non-human animal is a mammal (eg, a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal and / or a clone.

  Roughly: As used herein, the term “approximately” or “about” as applied to one or more predetermined values refers to a value that approximates the referenced value mentioned. In certain embodiments, “approximately” or “about” means in both directions (unless such number exceeds 100% of the possible value) unless otherwise specified or apparent from the context. Larger or smaller), 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10% of the referenced value mentioned, Refers to a range of values encompassed within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

  Biologically active substance: As used herein, the phrase “biologically active substance” refers to any substance that has activity in a biological system and / or organism. For example, when administered to an organism, a substance that has a biological effect on that organism is considered biologically active. In a specific embodiment, when a protein or polypeptide is biologically active, the portions of the protein or polypeptide that share at least one biological activity of the protein or polypeptide are generally “biological It is said to be a "logically active" moiety.

  Botulinum toxin: As used herein, the term “botulinum toxin” refers to any neurotoxin produced by Clostridium botulism. Unless otherwise specified, the term encompasses such neurotoxin fragments or portions (eg, light and / or heavy chains) that retain appropriate activity (eg, muscle relaxant activity). As used herein, the phrase “botulinum toxin” encompasses botulinum toxin serotypes A, B, C, D, E, F and G. Botulinum toxin as used herein includes both botulinum toxin complexes (ie, 300, 600 and 900 kD complexes) and purified (ie, eg, isolated) botulinum toxins (ie, eg, about 150 kD). Is also included. “Purified botulinum toxin” is defined as a botulinum toxin that has been isolated or substantially isolated from other proteins, including proteins of the botulinum toxin complex. The purified toxin may be more than 95% pure, preferably more than 99% pure. One skilled in the art will appreciate that the present invention is not limited to any particular source of botulinum toxin. For example, the botulinum toxin used in the present invention may be isolated from Clostridium botulinum, chemically synthesized, or produced by recombinant technology (ie, a host cell or organism other than Clostridium botulinum). (In).

  Characteristic part: As used herein, the phrase “characteristic part” of a substance corresponds in a broadest sense to some degree of sequence and / or structural identity and / or at least one functional characteristic. It is shared with the intact substance. For example, a “characteristic portion” of a protein or polypeptide is one that comprises a stretch of contiguous amino acids, or a stretch of contiguous amino acids, which together are a characteristic of a protein or polypeptide. In some embodiments, each such successive run generally comprises at least 2, 5, 10, 15, 20 or more amino acids. In general, a characteristic part is one that shares at least one functional characteristic with the corresponding native protein in addition to the sequence identity defined above. In some embodiments, the characteristic portion may be biologically active.

  Hydrophilic: As used herein, a “hydrophilic” material is a material that is soluble in a polar solvent. In some embodiments, the hydrophilic material can be temporarily combined with a polar solvent. In some embodiments, the hydrophilic material can temporarily bind to the polar solvent with hydrogen bonds. In some embodiments, the polar solvent is water. In some embodiments, the hydrophilic material may be ionic. In some embodiments, the hydrophilic material may be nonionic. In some embodiments, the hydrophilic material can be more easily dissolved in water, polar solvent or hydrophilic solvent than oil, non-polar solvent or hydrophobic solvent. In some embodiments, the hydrophilic material does not dissolve as easily in oil, non-polar or hydrophobic solvents as in water, polar or hydrophilic solvents. In some embodiments, one substance is more hydrophilic than another because it is more soluble in water, polar solvents, or hydrophilic solvents than other substances. In some embodiments, one substance is more hydrophilic than another because it is not as soluble in oils, non-polar solvents or hydrophobic solvents as other substances.

  Hydrophobic: As used herein, a “hydrophobic” material is a material that is soluble in a nonpolar solvent. In some embodiments, the hydrophobic material is repelled from the polar solvent. In some embodiments, the polar solvent is water. In some embodiments, the hydrophobic material is non-polar. In some embodiments, the hydrophobic material can be more easily dissolved in oil, nonpolar solvent or hydrophobic solvent than water, polar solvent or hydrophilic solvent. In some embodiments, the hydrophobic material does not dissolve as easily in water, polar solvent or hydrophilic solvent as oil, nonpolar solvent or hydrophobic solvent. In some embodiments, one substance is more hydrophobic than another because it is more soluble in oils, nonpolar solvents or hydrophobic solvents than other substances. In some embodiments, one substance is more hydrophobic than another because it is not as soluble in water, polar solvents or hydrophilic solvents as other substances.

  In connection with: As used herein, the phrase “delivering with” refers to the simultaneous delivery of two or more substances or agents. Specifically, according to the present invention, this phrase is used herein in the context of delivering the biologically active agents of the present invention with the nanoparticles and / or nanoparticle compositions. The substance or agent is either encapsulated in the nanoparticle and / or completely surrounded by the nanoparticle; the substance or agent is combined with the nanoparticle and / or nanoparticle composition; Delivered with the nanoparticles when embedded; and / or associated with the outer surface of the nanoparticle micelle membrane. The substance or agent delivered with the nanoparticle and / or nanoparticle composition may or may not be covalently bound to the nanoparticle and / or nanoparticle composition. The substance or agent delivered with the nanoparticle and / or nanoparticle composition of the present invention may or may not be associated with the nanoparticle and / or nanoparticle composition with an adsorptive force.

  Isolated: As used herein, the term “isolated” refers to (1) the components that are associated with it when it is first produced (naturally and / or in experimental equipment). Substances and / or entities separated from at least a portion and / or made, prepared and / or manufactured by human hands. An isolated substance and / or entity is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% of the other components with which it is originally associated. %, About 80%, about 90% or more. In some embodiments, the isolated material and / or entity has a purity greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. is there.

  Microfluidized: As used herein, the term “microfluidized” means subjecting to high shear forces. In some embodiments, such exposure to high shear forces is achieved by exposure to high pressure. In some embodiments, such high pressure is in the range of about 15,000 to about 26,000 psi. In some embodiments, such exposure to high shear forces is achieved by cavitation. In some embodiments, such exposure to high shear forces may cause the sample to produce a sample, eg, Microfluidizer® (Microfluidics Corporation / MFIC Corporation), or other similar useful for producing a uniform nanoparticle composition. This is achieved by passing it through a device such as a device. In some embodiments of the invention, the sample is microfluidized by exposure to high shear forces for a period of less than about 10 minutes. In some embodiments, the period is less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. In some embodiments, the period is in the range of about 1-2 minutes. In some embodiments, the period is about 30 seconds. In some embodiments of the present invention, the sample is “microfluidized” by a single exposure to high shear forces. Such an embodiment is referred to as “single pass” microfluidization.

  Nanoparticle: As used herein, the term “nanoparticle” refers to any particle having a diameter of less than 1000 nm (nm). In some embodiments, the nanoparticles have a diameter of less than 300 nm as defined by the National Science Foundation. In some embodiments, the nanoparticles have a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, the nanoparticle is a micelle in that it comprises a sealed compartment that is separated from the bulk solution by a micelle membrane. A “micellar membrane” includes an amphiphilic entity that aggregates to surround and seal (eg, define a lumen) a space or compartment.

Nanoparticle composition: As used herein, the term “nanoparticle composition” refers to any substance comprising at least one nanoparticle. In some embodiments, the nanoparticle composition is a uniform collection of nanoparticles. In some embodiments, the nanoparticle composition is a dispersion or emulsion. Generally, a dispersion or emulsion is formed when at least two immiscible materials are mixed. An “oil-in-water” dispersion is one in which oily particles (or hydrophobic or nonpolar) are dispersed in an aqueous dispersion medium. A “water-in-oil” dispersion is one in which aqueous (or hydrophilic or polar) particles are dispersed in an oil-based dispersion medium. Those skilled in the art will appreciate that the dispersion can be formed from any two immiscible media and is not strictly limited to a combination of aqueous and oily media. Thus, the term “dispersion medium” applies broadly to any dispersion medium, although it generally refers to the “aqueous” and “oily” categories. In some embodiments, the nanoparticle composition is a nanoemulsion. In some embodiments, the nanoparticle composition comprises micelles. In some specific embodiments, the nanoparticle composition is a PCT application number PCT / US07 / filed November 30, 2007. , Amphiphilic entity nanoparticles described in the title “AMPHIPHILIC ENTITY NANOPARTICLES”, which is incorporated herein by reference. In some embodiments, the nanoparticle composition is stable. In some embodiments, the nanoparticle composition comprises one or more biologically active agents that are delivered with the nanoparticles.

  Nutritional supplement: As used herein, the term “nutrient supplement” refers to any substance that is believed to provide a medical, health or biological benefit. In some embodiments, the nutritional supplement prevents the disease. In some embodiments, the nutritional supplement can provide a basic nutritional value. In some embodiments, the nutritional supplement is a food product or part of a food product. In some embodiments, the nutritional supplement is a category of isolated nutrients, dietary supplements, vitamins, minerals, herbs, fortified foods, healing foods, genetically modified foods and processed foods It may be. Nutritional supplements are also known as “phytochemical foods” or “functional foods”.

Premix: As used herein, the term “premix” is any combination of ingredients that are subsequently used to produce a nanoparticle composition according to the present invention. For example, a premix is any collection of components that, when subjected to high shear forces, produces nanoparticles according to the present invention. In some embodiments, the premix comprises two or more immiscible solvents. In some embodiments, the premix includes components that self-assemble into nanoparticles. In some embodiments, the premix includes components that self-assemble into micelles. In some embodiments, the premix is a PCT application number PCT / US07 / filed on Nov. 30, 2007. , Including one or more amphiphilic entities as described in the title “AMPHIFILIC ENTITY NANOPARTICLES”, which is incorporated herein by reference. In some embodiments, the premix comprises one or more unmodified peptides. In some embodiments, the premix includes at least one other biologically active agent. In some embodiments, the premix is agitated, mixed and / or stirred. In some embodiments, the premix is agitated, mixed and / or stirred before being subjected to high shear forces. In some embodiments, the premix includes at least one solubilizing component (ie, at least one component in solution). In some such embodiments, the premix is subjected to high shear forces after such solubilization is achieved.

  Pure: As used herein, a substance and / or entity is “pure” if it is substantially free of other ingredients. For example, a preparation containing more than about 90% of a particular substance and / or entity is generally considered to be a pure preparation. In some embodiments, the substance and / or entity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure.

Shear force: As used herein, the term “shear force” refers to a force parallel to a surface of a material that opposes a force normal to the surface of the material. In some embodiments, the composition is subjected to high shear forces to produce a uniform nanoparticle composition. Any method known in the art can be used to generate a high shear force. In some embodiments, cavitation is used to generate high shear forces. In some embodiments, high pressure homogenization is used to generate high shear forces. Alternatively or in addition, high shear forces can be applied at high pressures, eg, about 15,000 psi. In some embodiments, such high pressure is in the range of about 18,000 to about 26,000 psi. In some embodiments, it is in the range of about 20,000 to about 25,000 psi. In some embodiments, a high fluid force is generated using a Microfluidizer® Processor (Microfluidics Corporation / MFIC Corporation) or other similar device. Microfluidizer (R) processing equipment provides high pressure to scale down to the nanoscale region, and has microchannels (typically on the order of 75 microns) at high speeds (typically in the range of 50 m / s to 300 m / s) ) Accelerates the composition through a high shear rate. As the fluid exits the microchannel, it forms a jet stream that collides with a jet stream from the opposing microchannel. Within the channel, the fluid undergoes a high degree of shear (up to 10 ⁷ 1 / s) to a higher degree than in the prior art. Jet flow impingement results in sub-micron mixing. Therefore, in such a device, it is possible to achieve particle refinement and multiphase mixing with high shear and / or impact. In some embodiments of the invention, the sample is subjected to high shear forces for a period of less than about 10 minutes. In some embodiments, the period is less than about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 or about 1 minute. In some embodiments, the period is in the range of about 1 to about 2 minutes or less. In some embodiments, the period is about 30 seconds. In some embodiments of the invention, the sample is “microfluidized” by a single exposure to high shear forces. Such an embodiment is referred to herein as “single pass” microfluidization.

  Small molecule: In general, a “small molecule” is understood in the art as an organic molecule having a size of less than about 5 kilodaltons (Kd). In some embodiments, the small molecule is less than about 3 Kd, about 2 Kd or about 1 Kd. In some embodiments, the small molecule is less than about 800 Daltons (D), about 600D, about 500D, about 400D, about 300D, about 200D or about 100D. In some embodiments, the small molecule is non-polymeric. In some embodiments, the small molecule is not a protein, peptide or amino acid. In some embodiments, small molecules are not nucleic acids or nucleotides. In some embodiments, the small molecule is not a saccharide or polysaccharide.

  Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition of the invention can be administered, eg, for experimental, diagnostic, prophylactic and / or therapeutic purposes. . Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates and humans; insects; insects; etc.).

  Substantially: As used herein, the term “substantially” refers to a qualitative state that exhibits a predetermined feature or characteristic in all or nearly all degrees or degrees. An engineer in the field of biological technology does not know that a biological and chemical phenomenon can reach to completion and / or proceed completely or reach an absolute result or avoid it. It will be understood that, if at all, it is extremely rare. Thus, the term “substantially” is used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena.

  Stable: The term “stable”, as applied herein to a nanoparticle composition, indicates that the composition has one or more aspects of its physical structure over a period of time (eg, the particle size range of the particles And / or distribution). In some embodiments of the present invention, the stable nanoparticle composition may comprise an average particle size, a maximum particle size, a particle size range and / or a particle size distribution (ie, particles exceeding a specified particle size and / or Or the proportion of particles outside the specified particle size range) over a period of time. In some embodiments, the period is at least about 1 hour. In some embodiments, the period is about 5 hours, about 10 hours, about 1 day, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, About 6 months, about 8 months, about 10 months, about 12 months, about 24 months or more. In some embodiments, the period is in the range of about 1 day to about 24 months, about 2 weeks to about 12 months, about 2 months to about 5 months, and the like. For example, if the nanoparticle composition is subjected to long-term storage, temperature changes and / or pH changes, the majority of the nanoparticles in the population maintain a diameter within the indicated range (ie, for example, about 10 nm to 120 nm) The nanoparticle composition is stable. For some such populations, the majority is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99 %, About 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9% or more. In some embodiments of the invention, when the nanoparticle composition comprises one or more biologically active agents (eg, unmodified peptides), for a specified period of time and under a specified set of conditions Thus, if the concentration of the biologically active substance in the composition is maintained, the nanoparticle composition is considered stable.

  Substantially free: When about 50% or less of the particles in the composition have a diameter outside a certain range, the nanoparticle composition of the present invention will “substantially” particles outside that indicated range in diameter. Is not included. " In some embodiments, 25% or less of the particles are outside that range. In some embodiments, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5% %, 4%, 3%, 2%, 1%, 0.5% or less of the particles have a diameter outside the indicated range.

  Affected: An individual “affected” by a disease, disorder or condition (eg, facial wrinkles) has been diagnosed with or exhibits symptoms of the disease, disorder or condition.

  Therapeutically effective amount: As used herein, the term “therapeutically effective amount” is used to treat a disease, disorder and / or condition when administered to a patient suffering from or susceptible to a disease, disorder and / or condition. Means a sufficient amount of the nanoparticle composition of the present invention.

  Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. Point to.

  Treatment: As used herein, the term “treatment” (and also “treat” or “treating”) refers to one or more of a particular disease, disorder, and / or condition. Biological that partially or completely alleviates, ameliorates, revitalizes, stops, delays its onset, reduces its severity and / or reduces its incidence Refers to any administration of the active substance. Such treatment may be treatment of a subject that does not show signs of the disease, disorder, and / or condition involved and / or treatment of a subject that shows very early signs of the disease, disorder, and / or condition. Good. Alternatively or in addition, such treatment may be treatment of a subject who exhibits one or more definitive signs of the disease, disorder and / or condition involved.

  Toxic solvent: As used herein, the term “toxic solvent” refers to any substance that can alter, disrupt, remove, or destroy animal tissue. As will be appreciated by those skilled in the art, animal tissue can include live cells, dead cells, extracellular matrix, cellular junctions, biomolecules, and the like. To name a few, toxic solvents include dimethyl sulfoxide, dimethylacetimide, dimethylformamide, chloroform, tetramethylformamide, acetone, acetate ester and alkali.

  Uniform: As used herein with respect to nanoparticle compositions, the term “uniform” refers to a nanoparticle composition in which individual nanoparticles have a particle size size in a specified range. For example, in some embodiments, the uniform nanoparticle composition has a difference between a maximum diameter and a minimum diameter of about 600, about 550, about 500, about 450, about 400, about 350, about 300, about A composition that does not exceed 250, about 200, about 150, about 100, about 90, about 80, about 70, about 60, about 50 nm or less. In some embodiments, particles (eg, particles comprising an unmodified peptide) in a uniform nanoparticle composition of the invention have about 600, about 550, about 500, about 450, about 400, about 350, about It has a diameter less than or less than 300, about 250, about 200, about 150, about 130, about 120, about 115, about 110, about 100, about 90, about 80 nm. In some embodiments, particles (eg, particles comprising an unmodified peptide) in a uniform nanoparticle composition of the invention have a diameter in the range of about 10 to about 600 nm. In some embodiments, particles (eg, particles comprising an unmodified peptide) in a uniform nanoparticle composition of the invention have about 10 to about 300, about 10 to about 200, about 10 to about 150, about 10 to about 130, about 10 to about 120, about 10 to about 115, about 10 to about 110, about 10 to about 100, or about 10 to about 90 nm in diameter. In some embodiments, particles (eg, particles comprising unmodified peptides) in the botulinum nanoparticle compositions of the present invention have about 300, about 250, about 200, about 150, about 130, about 120, about 115. , Having an average particle size below about 110, about 100 or about 90 nm. In some embodiments, the average particle size is in the range of about 10 to about 300, about 50 to about 250, about 60 to about 200, about 65 to about 150, about 70 to about 130 nm. In some embodiments, the average particle size is about 80 to about 110 nm. In some embodiments, the average particle size is about 90 to about 100 nm. In some embodiments, the majority of particles (eg, particles comprising an unmodified peptide) in a uniform nanoparticle composition of the present invention have a diameter that is below a specified size or within a specified range. Have In some embodiments, the majority is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% in the composition, There are more than 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of the particles. In some embodiments of the invention, a uniform nanoparticle composition is achieved by microfluidizing a sample. In some embodiments of the invention, uniform nanoparticle compositions are prepared by exposure to high shear forces, for example by microfluidization.

Unmodified peptide: As used herein, the term “unmodified peptide” refers to a peptide that has not been chemically modified by the addition of other covalently attached functional groups to achieve transdermal delivery of the peptide. Point to. In some embodiments, the peptide has not undergone chemical modifications that add pendant acetyl and / or palmitoyl groups. In some embodiments, the peptide has not undergone chemical modifications that add any functional pendant groups.
Nanoparticles As discussed herein, the present invention provides nanoparticle compositions comprising one or more unmodified peptides. In some embodiments, such nanoparticle compositions further comprise one or more biologically active agents in addition to the unmodified peptide. In some embodiments, the nanoparticle composition is formulated with one or more other ingredients, eg, in the preparation of a pharmaceutical or cosmetic product. In some embodiments, such pharmaceutical or cosmetic preparations are formulated to achieve transdermal delivery of the unmodified peptide (and / or one or more biologically active substances).

  In some embodiments, the nanoparticle compositions of the present invention are stable. In some embodiments, the nanoparticle composition is uniform.

  In some embodiments, the uniform nanoparticle composition has a difference between a minimum diameter and a maximum diameter of about 600 nm, about 550 nm, about 500 nm, about 450 nm, about 400 nm, about 350 nm, about 300 nm, about 250 nm, about Includes populations of particles not exceeding 200 nm, about 150 nm, or about 100 nm.

  In some embodiments, the nanoparticles of the present invention have about 1000, about 600, about 550, about 500, about 450, about 400, about 350, about 300, about 250, about 200, about 150, about 130, About 120, about 115, about 110, about 100, about 90, about 80, having a diameter less than or less than about 50 nm.

  In some embodiments, the nanoparticles of the invention have 1 nm to 1000 nm, 1 nm to 600 nm, 1 nm to 500 nm, 1 nm to 400 nm, 1 nm to 300 nm, 1 nm to 200 nm, 1 nm to 150 nm, 1 nm to 120 nm, 1 nm to 100 nm, It has a diameter of 1 nm to 75 nm, 1 nm to 50 nm, or 1 nm to 25 nm. In some embodiments, the nanoparticles of the present invention have a diameter of 1 nm to 15 nm, 15 nm to 200 nm, 25 nm to 200 nm, 50 nm to 200 nm, or 75 nm to 200 nm.

  In some embodiments, the total particle distribution includes a particle size size within a specified range. In some embodiments, less than 50%, 25%, 10%, 5%, or 1% of the total particle distribution is outside the specified range of particle size sizes. In some embodiments, less than 1% of the total particle distribution is outside the specified range of particle size sizes. In certain embodiments, the nanoparticle composition is substantially free of particles having a diameter greater than 300 nm, 250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm, 50 nm, or 25 nm.

  In some embodiments, the nanoparticles in the nanoparticle composition of the invention have about 300 nm, about 250 nm, about 200 nm, about 150 nm, about 130 nm, about 120 nm, about 115 nm, about 110 nm, about 100 nm, about 90 nm or It has an average particle size below about 50 nm. In some embodiments, the average particle size is in the range of about 10 nm to about 300 nm, 50 nm to about 250 nm, 60 nm to about 200 nm, 65 nm to about 150 nm, or 70 nm to about 130 nm. In some embodiments, the average particle size is from about 80 nm to about 110 nm. In some embodiments, the average particle size is about 90 to about 100 nm.

  In some embodiments, the nanoparticle compositions of the present invention are substantially free of particles having a diameter greater than 300 nm. Specifically, in some embodiments, less than 50% of the nanoparticles in the nanoparticle composition of the present invention have a diameter greater than 300 nm. In some embodiments, less than 25% of the particles have a diameter greater than 300 nm. In some embodiments, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5% %, 4%, 3%, 2%, 1%, less than 0.5% or less of the particles have a diameter greater than 300 nm. Further, in some embodiments, the nanoparticles in the nanoparticle composition of the present invention have a diameter in the range of 10 nm to 300 nm.

  In some embodiments, the nanoparticle compositions of the present invention are substantially free of particles having a diameter greater than 200 nm. Specifically, in some embodiments, less than 50% of the nanoparticles in the nanoparticle composition of the invention have a diameter greater than 200 nm. In some embodiments, less than 25% of the particles have a diameter greater than 200 nm. In some embodiments, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5% %, 4%, 3%, 2%, 1%, less than 0.5% or less of the particles have a diameter greater than 200 nm. Further, in some embodiments, the nanoparticles in the nanoparticle composition of the present invention have a diameter in the range of 10 nm to 200 nm.

  In some embodiments, the nanoparticle compositions of the present invention are substantially free of particles having a diameter greater than 120 nm. Specifically, in some embodiments, less than 50% of the nanoparticles in the nanoparticle composition of the present invention have a diameter greater than 120 nm. In some embodiments, less than 25% of the particles have a diameter greater than 120 nm. In some embodiments, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5% %, 4%, 3%, 2%, 1%, less than 0.5% or less of the particles have a diameter greater than 120 nm. Further, in some embodiments, the nanoparticles in the nanoparticle composition of the present invention have a diameter in the range of 10 nm to 120 nm.

  In some embodiments, the majority of the nanoparticles in the composition of the invention have a diameter that is less than a specified size or within a specified range. In some embodiments, the majority is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% of the particles in the composition. 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more.

  The zeta potential is the magnitude of the potential at the shear plane. The shear plane is a virtual layer that separates a thin layer at the liquid boundary from a solid surface (for example, the surface of the nanoparticle of the present invention) and exhibits elastic behavior by a liquid that exhibits normal viscous behavior (for example, a liquid dispersion medium). The surface. In some embodiments, inventive nanoparticles have a zeta potential in the range of −50 mV to +50 mV. In some embodiments, inventive nanoparticles have a zeta potential in the range of −25 mV to +25 mV. In some embodiments, inventive nanoparticles have a zeta potential in the range of −10 mV to +10 mV.

  In some embodiments, the nanoparticle composition of the present invention is an emulsion or dispersion. In general, an emulsion or dispersion is formed from at least two immiscible materials, one of which is a dispersion medium in which particles (eg, nanoparticles comprising a “dispersed medium”) are dispersed (ie, , Liquid medium). An “oil-in-water” dispersion is one in which oily particles are dispersed in an aqueous dispersion medium. A “water-in-oil” dispersion is one in which aqueous particles are dispersed in an oil-based dispersion medium. One skilled in the art will appreciate that the dispersion can be formed from any two immiscible media and is not strictly limited to a combination of aqueous and oily media. Thus, the term “dispersion medium” applies broadly to any dispersion medium, although it generally refers to the categories “aqueous” and “oily”. For example, an emulsion or dispersion is a series of immiscible materials composed of hydrophobic / hydrophilic materials; polar / nonpolar materials, etc., regardless of whether such materials are strictly “aqueous” or “oily”. It can be prepared from a functional material.

  In some embodiments, the nanoparticle compositions of the present invention comprise a micelle structure (eg, the nanoparticles are micelles). In some embodiments, such micelle structures are cross-linked. In some embodiments, such micelle structures are not cross-linked.

  In some embodiments, the nanoparticle compositions of the present invention self-assemble from the combined component assembly. In some embodiments, inventive nanoparticle compositions are prepared by subjecting a combination of ingredients (ie, a “premix”) to high shear forces. In some embodiments, the high shear force is applied by high pressure, cavitation, homogenization and / or microfluidization. In some embodiments, the combined nanoparticle-forming components are agitated, stirred or mixed. In some such embodiments, the components are subjected to high shear forces after mixing. In some specific embodiments, the mixing can be performed over a period of, for example, less than 1 hour, or less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 hours. In some embodiments, solubilization is performed.

  In some embodiments of the present invention, the production of the nanoparticle composition includes, for example, dialysis of a component assembly to remove any organic solvent and / or lyophilization to make the composition.

  It should be understood that in some embodiments of the present invention using a premix, the premix components aggregate into particles prior to exposure to high shear forces. At least some of such particles may be microparticles or nanoparticles. In some embodiments, the nanoparticle compositions of the present invention are prepared from a premix selected from the group comprising a suspension or microemulsion. However, in some embodiments, no particle structure is formed in the premix prior to exposure to high shear forces.

  In some embodiments of the invention, all of the components present in the final nanoparticle composition are present in the premix and are subjected to high shear forces to form the nanoparticle composition. In some embodiments of the invention, one or more of the components present in the final nanoparticle composition are missing from the premix or are present in the premix in lesser amounts than in the final nanoparticle composition. . That is, in some embodiments of the invention, one or more materials are added to the nanoparticle composition after applying high shear stress to the premix.

  In certain embodiments of the invention, the premix is prepared as a solution prior to exposure to high shear forces. Specifically, for nanoparticle compositions that include at least one biologically active agent (eg, an unmodified peptide), the biologically active agent is dissolved in the premix prior to exposure to high shear stress. It is often desirable to do so. Thus, in many embodiments, the biologically active agent is soluble in at least one of the media (or with the media used in the premix). In some embodiments of the invention, such melting requires heating. In other embodiments, it is not required.

  In some embodiments of the invention, the nanoparticle composition comprises one or more aqueous, polar or hydrophilic media, one or more oily, nonpolar or hydrophobic media, one or more Prepared from components comprising micelle components, one or more surfactants or emulsifiers, one or more biologically active substances and / or one or more release retardants, and the like.

  Those skilled in the art will be well aware of suitable aqueous media that can be used as a dispersion medium or as a dispersion medium according to the present invention. Typical such aqueous media include, for example, water, saline (including phosphate buffered saline), water for injection, short chain alcohol, 5% dextrose, Ringer's solution (Lactated Ringer's solution, Lactated Ringer's solution +5 % Dextrose injection, acylated Ringer's injection), Normosol-M, Isolyte E and the like and combinations thereof.

Those skilled in the art will also be well aware of suitable oily media that can be used as a dispersion medium or as a medium to be dispersed according to the present invention. In some embodiments, the oil can include one or more fatty acid groups or salts thereof. In some embodiments, the fatty acid group can include a digestible long chain (eg, C ₈ -C ₅₀ ), substituted or unsubstituted hydrocarbon. In some embodiments, the fatty acid group may be a C ₁₀ -C ₂₀ fatty acid or salt thereof. In some embodiments, the fatty acid group may be a C ₁₅ -C ₂₀ fatty acid or salt thereof. In some embodiments, the fatty acid group may be a C ₁₅ -C ₂₅ fatty acid or salt thereof. In some embodiments, the fatty acid group may be unsaturated. In some embodiments, the fatty acid group may be monounsaturated. In some embodiments, the fatty acid group may be polyunsaturated. In some embodiments, the double bond of the unsaturated fatty acid group may be a cis structure. In some embodiments, the double bond of the unsaturated fatty acid group may be a trans structure.

  In some embodiments, the fatty acid group is one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid or lignoceric acid. Good. In some embodiments, the fatty acid group is palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid or elca It may be one or more of acids.

  In some embodiments, the oil is a liquid triglyceride. In certain embodiments, the oil is medium chain (eg, 6-12 carbon) triglycerides (eg, Labrafac WL1349, coconut oil, palm kernel oil, camphor kernel oil, etc.). In certain embodiments, the oil is a short chain (eg, 2-5 carbon) triglyceride. In certain embodiments, the oil is a long chain (eg, greater than 12 carbons) triglyceride (eg, soybean oil, sunflower oil, etc.).

  Oils suitable for use in the present invention include, but are not limited to, almond, apricot, avocado, babas, bergamot, black currant seed, borage, cadet, chamomile, canola, caraway, carnauba, castor, cinnamon , Cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui Fruit, lavandin, lavender, lemon, lychee aqaba, macadamia nut, aoi, mango seed, meadowform seed, mineral, mink, nutmeg, olive, orange, orange raffie, palm, palm kernel, peach nut, peanut, poppy seed, Pumpkin Rapeseed, rice bran, rosemary, safflower, sandalwood, sasanqua, savory, sea buckthorn, sesame, shea butter, silicone, soy, sunflower, tea tree, thistle, camellia, vetiver, walnut, wheat germ oil and mixtures thereof Is included. Synthetic oils suitable for use in the present invention include, but are not limited to, caprylic / capric triglycerides, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, octyldodecanol, oleyl alcohol and combinations thereof Is included.

  Suitable micelle components can include, for example, one or more amphiphilic entities. Useful amphiphilic entities include natural entities, synthetic entities, and entities that contain both natural and synthetic components. In some embodiments, an amphiphilic entity can include one or more polymers and / or one or more compounds having polymer characteristics.

  In general, an amphiphilic entity is one that is both hydrophobic and hydrophilic. As those skilled in the art will appreciate, amphiphilic entities can be configured in many different ways. In some embodiments, an amphiphilic entity can include one or more individual compounds or substances that are themselves amphiphilic. To name a few, such compounds or molecules include polyethylene glycol (PEG), phospholipids, cholesterol, glycolipid fatty acids, bile acids and saponins. PEG has been recognized by the US Food and Drug Administration as being generally safe for use in food, cosmetics and pharmaceuticals. PEG is water soluble, non-toxic, odorless, lubricious, non-volatile and non-irritating.

  In some embodiments, an amphiphilic entity may include one or more individual components that are not amphiphilic per se but have some hydrophilic or hydrophobic character. In such embodiments, two or more such non-amphiphilic components are generally associated with each other so that the collection of individual components is amphiphilic. Such an association may or may not include a covalent bond. Such associations may include non-covalent bonds (eg, due to electrostatic interactions, affinity interactions, hydrophobic interactions, hydrogen bonds, van der Waals interactions, ionic interactions, dipole-dipole interactions, etc.) it can. In general, such association can include any associated force, binding, or attachment means.

  In some embodiments, the amphiphilic entity used in the present invention can be composed of two or more individual components having different degrees of hydrophilicity or hydrophobicity. In certain embodiments, the amphiphilic entity can include at least one hydrophilic component and at least one hydrophobic component. In certain embodiments, the “hydrophilic” and “hydrophobic” components are hydrophilic or hydrophobic with respect to each other.

  In some embodiments, two or more components of different degrees of hydrophilicity or hydrophobicity can be joined together by covalent bonds to form a homopolymer or copolymer. In some embodiments, the copolymer may be a block copolymer. In some embodiments, the copolymer may be a graft copolymer.

  In some embodiments, the amphiphilic entity may comprise or consist of an amphiphilic block copolymer. In some embodiments, the amphiphilic block copolymer may be a diblock copolymer. In certain embodiments, the amphiphilic diblock copolymer can include a first polymer block and a second polymer block covalently linked at the chain ends. In certain embodiments, the first polymer block can include repeating units of the hydrophilic component and the second polymer block can include repeating units of the hydrophobic component. In certain embodiments, the first polymer block can include a repeating unit of a hydrophobic component and the second polymer block can include a repeating unit of a hydrophilic component. In some embodiments, the amphiphilic block copolymer may be a multi-block copolymer. In certain embodiments, the amphiphilic block copolymer can comprise a plurality of alternating blocks consisting of two or more polymers covalently linked at the chain ends. In certain embodiments, the amphiphilic block copolymer can include a plurality of alternating hydrophilic and hydrophobic blocks covalently bonded at the chain ends. In certain embodiments, each block of alternating blocks can include a repeating unit consisting of either a hydrophilic component or a hydrophobic component.

  In some embodiments, the amphiphilic entity may comprise or consist of an amphiphilic graft copolymer. In some embodiments, the amphiphilic graft copolymer may comprise or consist of a block of polymer covalently bonded to a side chain of another block of the polymer. In certain embodiments, each polymer block may comprise or consist of repeating units of hydrophilic or hydrophobic components. In certain embodiments, the amphiphilic graft copolymer may comprise or consist of a first polymer block and a second polymer block covalently bonded to a side chain of the first polymer block. In certain embodiments, the first polymer block can comprise or consist of repeating units of the hydrophilic component and the second block can comprise repeating units of the hydrophobic component. In certain embodiments, the first polymer block can comprise or consist of repeating units of a hydrophobic component and the second block can comprise repeating units of a hydrophilic component.

  In some embodiments, the amphiphilic block or graft copolymer may comprise a hydrophilic polymer block comprising a polysaccharide repeating unit and a hydrophobic polymer block comprising a polyester or polysaccharide repeating unit. Alternatively or in addition, the amphiphilic block or graft copolymer may comprise a hydrophobic polymer block comprising polysaccharide repeating units and a hydrophilic polymer block comprising polyester or polysaccharide repeating units. Such hydrophilic polymer blocks can include repeating units of any type of hydrophilic polymer such as polysaccharides (eg, pullulan) or polyalkene oxides (eg, polyethylene oxide). The hydrophobic polymer block can comprise repeating units of any type of hydrophobic polymer, such as polycaprolactone or polyamide (eg, polycaprolactam).

  In some embodiments, the hydrophilic portion of the amphiphilic entity may be nonionic. In some embodiments, the hydrophilic component of the amphiphilic entity includes one or more ionic groups. In general, such ionic groups are hydrophilic and can impart hydrophilic properties to the amphiphilic entity.

In some embodiments, the ionic group may be cationic. In some embodiments, the cationic group is ammonium (NH ₄ ⁺ ), nitronium (NO ₂ ⁺ ), nitrosyl (NO ⁺ ), hydronium (H ₃ O ⁺ ), mercuric (Hg ₂ ²⁺ ), phosphonium ( PH ₄ ⁺ ), vanadyl (VO ²⁺ ), or a salt thereof.

In some embodiments, the ionic group may be anionic. In some embodiments, the anionic group is a fatty acid, arsenide (As 3− ), azide (N 3 − ), bromide (Br − ), chloride (Cl − ), fluoride (F − ), hydride (H − ). , Iodide (I − ), nitride (N 3− ), oxide (O 2− ), phosphide (P 3− ), selenide (Se 2− ), sulfide (S 2− ), peroxide (O 2 2− ), Arsenic acid (AsO 4 3− ), Arsenous acid (AsO 3 3− ), Boric acid (BO 3 3− ), Perbromic acid (BrO 4 − ), Bromic acid (BrO 3 − ), Bromic acid (BrO) 2 -), hypobromite (BrO -), carbonate (CO 3 2-), bicarbonate (HCO 3 -), chlorate (ClO 3 -), perchlorate (ClO 4 -), chlorite ( ClO 2 -), hypochlorite (ClO -), click Beam acid (CrO 4 2-), dichromate (Cr 2 O 7 2-), perfluorinated acid (BrO 4 -), fluorinated acid (BrO 3 -), nitrite fluorine acid (BrO 2 -), hypofluorous Acid (BrO − ), periodic acid (IO 4 − ), iodic acid (IO 3 − ), iodic acid (IO 2 − ), hypoiodous acid (IO − ), nitric acid (NO 3 − ), nitrous acid (NO 2 − ), phosphoric acid (PO 4 3− ), hydrogen phosphate (HPO 4 2− ), dihydrogen phosphate (H 2 PO 4 − ), phosphorous acid (PO 3 3− ), silicic acid ( SiO 3 2− ), sulfuric acid (SO 4 2− ), thiosulfuric acid (S 2 O 3 2− ), hydrogen sulfate (HSO 4 − ), sulfurous acid (SO 3 2− ), hydrogen sulfite (HSO 3 − ), sulfone acid (-S (-O) 2 -O - ), acetic acid (C 2 H 3 O 2 - ), formic acid (HCO 2 -) Oxalic acid (C 2 O 4 2− ), hydrogen oxalate (HC 2 O 4 − ), citric acid (C 6 H 5 O 7 3− ), succinic acid (C 4 H 4 O 4 2− ), fumaric acid Acid (C 4 H 2 O 4 2− ), Malic acid (C 4 H 5 O 5 2− ), Hydrogen sulfide (HS − ), Telluride (Te 2− ), Amide (NH 2 − ), Cyanic acid (OCN) -), thiocyanate (SCN -), cyanide (CN -), hydroxide (OH -), permanganate (MnO 4 -), or a salt thereof.

  In some embodiments, the hydrophilic component of the amphiphilic entity may comprise or consist of a nucleic acid. For example, the nucleic acid polymer can include DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid polymer may be an oligonucleotide and / or a polynucleotide. In some embodiments, the nucleic acid polymer is an oligonucleotide and / or modified oligonucleotide; antisense oligonucleotide and / or modified antisense oligonucleotide; cDNA; genomic DNA; viral DNA and / or RNA; DNA and / or RNA Chimeras; plasmids; cosmids; gene fragments; artificial chromosomes and / or natural chromosomes (eg, yeast artificial chromosomes) and / or parts thereof, RNA (eg, mRNA, tRNA, rRNA and / or ribozymes); peptide nucleic acids (PNA) A polynucleotide comprising a synthetic analogue of a nucleic acid, which may or may not be modified; in various structural forms including single-stranded DNA, double-stranded DNA, superhelical DNA and / or triple-helical DNA DNA Z-DNA; and / or a combination thereof.

  In some embodiments, the hydrophilic component of the amphiphilic entity may comprise or consist of a carbohydrate. In some embodiments, the carbohydrate may be a polysaccharide consisting of monosaccharides (or derivatives thereof) linked by glycosidic bonds, as is known in the art. Such sugars include, but are not limited to, glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucolonic acid, galactronic acid, mannuronic acid, glucosamine, galatosamine, and neuramin Contains acid. In some embodiments, the polymer may be a hydrophilic carbohydrate including aminated, carboxylated and sulfated polysaccharides. In some embodiments, the hydrophilic carbohydrate is pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxycellulose, methylcellulose, dextran, cyclodextran lan, glycogen, starch, hydroxyethyl starch, carrageenan, glycone, amylose, It may be one or more of chitosan, N, O-carboxymethylchitosan, algin and alginic acid, starch, chitin, heparin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan and xanthan. In some embodiments, hydrophilic polysaccharides can be modified to become hydrophobic by introducing multiple side chain hydrophobic groups. In some embodiments, hydrophobic carbohydrates include cellulose acetate, pullulan acetate, konjac acetate, amylose acetate and dextran acetate.

  In some embodiments, the hydrophilic component of the amphiphilic entity may comprise or consist of gums including, but not limited to, xanthan gum, alginic acid, karaya gum, sodium alginate, and / or locust bean gum. .

  In some embodiments, the components of the amphiphilic entity may comprise or consist of proteins. In some embodiments, the protein is the hydrophilic component of an amphiphilic entity. In other embodiments, the protein is a hydrophobic component of an amphiphilic entity. Examples of proteins that can be used in the present invention include, but are not limited to, albumin, collagen or poly (amino acids) (eg, polylysine).

In some embodiments, the hydrophobic component of the amphiphilic entity may comprise or consist of one or more fatty acid groups or salts thereof. In general, such groups are typically hydrophobic and can impart hydrophobic properties to the amphiphilic entity. In some embodiments, the fatty acid group can comprise a digestible long chain (eg, C ₈ -C ₅₀ ), substituted or unsubstituted hydrocarbon. In some embodiments, the fatty acid group may be a C ₁₀ -C ₂₀ fatty acid or salt thereof. In some embodiments, the fatty acid group may be a C ₁₅ -C ₂₀ fatty acid or salt thereof. In some embodiments, the fatty acid group may be a C ₁₅ -C ₂₅ fatty acid or salt thereof. In some embodiments, the fatty acid group may be unsaturated. In some embodiments, the fatty acid group may be monounsaturated. In some embodiments, the fatty acid group may be polyunsaturated. In some embodiments, the double bond of the unsaturated fatty acid group may be a cis structure. In some embodiments, the double bond of the unsaturated fatty acid group may be a trans structure.

  In some embodiments, the fatty acid group is one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid or lignoceric acid. Good. In some embodiments, the fatty acid group is palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, α-linoleic acid, γ-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or elca It may be one or more of acids.

  In some embodiments, the hydrophobic component of the amphiphilic entity includes, for example, polycarbonate (eg, poly (1,3-dioxane-2one)), polyanhydride (eg, poly (sebacate anhydride)), poly Hydroxy acids (eg poly (β-hydroxyalkanoates)), polypropyl fumerate, polycaprolactone, polyamides (eg polycaprolactam), polyacetals, polyethers (eg polylactides and polyglycolides), biodegradable poly It may comprise or consist of one or more biocompatible and / or biodegradable synthetic polymers including cyanoacrylate, polyvinyl alcohol and biodegradable polyurethane. For example, the amphiphilic entity includes the following biodegradable polymers: poly (lactic acid), poly (glycolic acid), poly (caprolactone), poly (lactide-co-glycolide), poly (lactide-co-caprolactone). , Poly (glycolide-co-caprolactone) and poly (DL-lactide-co-glycolide).

  In some embodiments, the hydrophobic component of the amphiphilic entity may comprise or consist of one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), methacrylic acid. Acid alkylamide copolymer, poly (methyl methacrylate), poly (methacrylic anhydride), methyl methacrylate, polymethacrylate, poly (methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymer and one or more of the above polymers Combinations containing are included. The acrylic polymer can comprise a fully polymerized copolymer of acrylic acid and methacrylic acid ester having a low content of quaternary ammonium groups.

  In some embodiments, the hydrophobic component of the amphiphilic entity may comprise or consist of a polyester. Examples of such polyesters include, for example, polyalkylene glycol, poly (glycolide-co-lactide), PEGylated poly (lactic acid-co-glycolic acid), poly (lactic acid), PEGylated poly (lactic acid), poly (glycolic acid) ), PEGylated poly (glycolic acid), copolymers of polylactic acid and polyglycolic acid, and derivatives thereof. In some embodiments, the polyester includes, for example, polyanhydride, poly (orthoester) PEGylated poly (orthoester), poly (caprolactone), PEGylated poly (caprolactone), polylysine, PEGylated polylysine, poly (ethylene). Imine), PEGylated poly (ethyleneimine) and derivatives thereof. In some embodiments, the polyester is, for example, polycaprolactone, poly (L-lactide-co-L-lysine), poly (serine ester), poly (4-hydroxy-L-proline ester), poly [α- (4-aminobutyl) -L-glycolic acid], and derivatives thereof.

  Suitable surfactants or emulsifiers include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholines (DPPC); dioleoylphosphatidylethanolamine (DOPE); Phosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; palmitic acid or olein Surface active fatty acids such as acids; fatty acids; fatty acid amides; sorbitan trioleate (Span 85) grease Sorbate monolaurate (Span 20); polysorbate 20 (Tween-20); polysorbate 60 (Tween-60); polysorbate 65 (Tween-65); polysorbate 80 (Tween-80); polysorbate 85 (Tween-85); Surfactin; poloxamer; sorbitan fatty acid esters such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; Dipalmitoyl phosphatidylglycerol; stearylamine; dodecylamine; Decyl - amine; acetyl palmitate; glycerol ricinoleate; hexadecyl stearate; tyloxapol; poly (ethylene glycol) 5000- phosphatidylethanolamine; poly (ethylene glycol) 400- monostearate; it includes and phospholipids. The surfactant component may be a mixture of different surfactants. These surfactants can be extracted from natural resources and purified, or they can be prepared synthetically in the laboratory. In a preferred embodiment, the surfactant is commercially available.

  In certain embodiments of the invention, the relative amounts of components used to prepare the nanoparticle composition of the invention are selected or adjusted to produce nanoparticles having the desired characteristics. In some embodiments, the oil and surfactant are used in a ratio ranging from 0.25 to 10. In some embodiments, the ratio of oil to surfactant is about 0.25: 1, about 0.5: 1, about 1: 1, about 2: 1, about 3: 1, about 4: 1, 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1 or about 10: 1. In some embodiments, the ratio of surfactant to oil is about 0.5: 1, about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6 :. 1, about 7: 1, about 8: 1, about 9: 1 or about 10: 1. In some embodiments, the oil and surfactant are used in a ratio ranging from 0.25 to 2. In some embodiments, the ratio of oil to surfactant is about 0.25: 1, about 0.5: 1, about 1: 1, or about 2: 1. In some embodiments, the surfactant to oil ratio is about 0.5: 1, about 1: 1, or about 2: 1. In certain embodiments, the ratio of oil to surfactant is approximately 1: 1.

  In some embodiments, the percentage of oil in the composition from which the nanoparticles are prepared (eg, in the premix) ranges from 0% to 30%. In some embodiments, the proportion of oil in the composition from which the nanoparticles are prepared (eg, in the premix) is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%. %, About 7%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, About 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29% or about 30%. In some embodiments, the percentage of oil is about 8%. In some embodiments, the percentage of oil is about 5%.

  In some embodiments using one or more amphiphilic entities, the proportion of amphiphilic entities in the composition from which the nanoparticles are prepared (eg, in the premix) is 40% to 99%, 50% % -99%, 60% -99%, 70% -99%, 80% -99%, 80% -90% or 90% -99%. In some embodiments, the percentage of amphiphilic entity in the composition from which the nanoparticles are prepared (eg, in the premix) is about 75%, about 76%, about 77%, about 78%, about 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% About 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%.

  The proportion of substances having surfactant activity in the premix may range from 0% to 99%, 10% to 99%, 25% to 99%, 50% to 99% or 75% to 99%. . In some embodiments, the percentage of surfactant-active material in the premix may range from 0% to 75%, 0% to 50%, 0% to 25%, or 0% to 10%. . In some embodiments, the proportion of surfactant in the composition from which the nanoparticles are prepared (eg, in the premix) ranges from 0% to 30%. In some embodiments, the surfactant percentage is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23% About 24%, about 25%, about 26%, about 27%, about 28%, about 29% or about 30%. In some embodiments, the surfactant percentage is about 8%. In some embodiments, the surfactant percentage is about 5%.

  In some embodiments, the nanoparticle composition does not include more than one oil. In some embodiments, the nanoparticle composition can include more than one oil. In some embodiments, the nanoparticle composition does not include more than one surfactant. In some embodiments, the nanoparticle composition can include two or more surfactants. In some embodiments, the nanoparticle composition is completely free of or substantially free of toxic ingredients.

  In some embodiments, the nanoparticle composition consists essentially of water, oil, surfactant, and at least one biologically active agent (eg, and unmodified peptide). In some embodiments, the nanoparticle composition comprises water, oil, surfactant, at least one biologically active material, and at least one material used to make and / or store the nanoparticle composition. Essentially consists of

In some embodiments, the nanoparticle composition consists essentially of water, oil, surfactant, and unmodified peptide. In some embodiments, the nanoparticle composition consists essentially of water, oil, surfactant, unmodified peptide, and at least one substance used to make and / or store the nanoparticles.
Unmodified Peptides Any of a variety of peptides can be incorporated into a nanoparticle composition according to the present invention. In most embodiments, the peptide is less than about 100 amino acids in length. In some embodiments, the peptide is about 90, about 80, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15. , About 13, about 12, about 10, about 9, about 8, about 7, about 6 or less than about 5 amino acids in length. In some specific embodiments, the peptide is a pentapeptide. In some embodiments, the peptide incorporated into the nanoparticle composition comprises only naturally occurring amino acids. In some embodiments, the peptide comprises one or more non-naturally occurring amino acids.

  The unmodified short peptides used according to the invention are generally those having biological activity in the skin (including epidermis and dermis), subcutaneous tissue (including adipose tissue) and / or adjacent muscles. Such peptides include, but are not limited to, peptides that promote extracellular matrix generation (eg, KTTKS, SEQ ID NO: 1; EYKTTKSSRL, SEQ ID NO: 2; VIEWKTKT, SEQ ID NO: 3; KTTK, SEQ ID NO: 4; 5; GKTVIEYKTKTKSRL, SEQ ID NO: 6; WGKTVIEYKTKTKSSRLPID, SEQ ID NO: 7; Peptides (eg, gastrin releasing peptide, VGVAPG, SEQ ID NO: 11; YYRADA, SEQ ID NO: 12; GHK, SEQ ID NO: 13, interferon, interferon Inducers), and peptides for treating excess accumulation of extracellular matrix resulting in conditions such as hypertrophic wounds, keloids, and localized or systemic sclerosis (scleroderma) (eg, P144; TSLDASIIWAMMQN (Katayama et al .; supra, Lupo, supra; Robinson et al., Supra; Bhartya et al., 1992, J. Cell. Physiol, 150: 312; and Santiago et al., 2005, J. Investigative. Dermatology, 125: 450; all of which are incorporated herein by reference). See Table 1 below which defines peptide abbreviations.

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Other Components As shown herein, the nanoparticle compositions of the present invention can comprise or be combined with one or more components. Specific examples of such other ingredients are discussed here.
Biologically active substances Any biologically active substance can be delivered according to the present invention including, for example, therapeutic agents, diagnostic agents, prophylactic agents, nutritional agents, cosmetics, and / or dermatological agents. Such biologically active substances include small molecules, organometallic compounds, nucleic acids, proteins (including multimeric proteins, protein complexes, etc.), peptides, lipids, carbohydrates, herbs, hormones, metals, radioactive elements and compounds, drugs, It may be a vaccine, an immunizing agent, etc., and / or combinations thereof. Such a biologically active substance may be encapsulated in the micellar membrane of the nanoparticles of the present invention, adsorbed on its surface, or present at its interface and / or within it.

  In some embodiments, the proportion of biologically active agent in the composition used to prepare the nanoparticles of the invention (eg, in the premix) and / or in the nanoparticles ranges from 0.1% to 25 % Range. In some embodiments, the percentage of biologically active agent is 0.1% to 20%, 0.1% to 15%, 0.1% to 10%, 0.1% to 5%, or 0.1 % To 1%. In some embodiments, the percentage of biologically active agent ranges from 1% to 20%, 5% to 20%, 10% to 20%, 15% to 20%, or 15% to 25%. In some embodiments, the percentage of biologically active agent is 0.1%. In some embodiments, the percentage of biologically active agent is greater than 25%. In some embodiments, the percentage of biologically active agent is about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%. About 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25% or more.

  The biologically active substance involved can be made or obtained by any possible method or approach. The biologically active agent can include or be modified to include one or more moieties to facilitate use or delivery of the nanoparticle with the present invention. Such modifications should not interfere with the biological activity of the drug. In some embodiments, the modification can optionally be removed in vivo. For example, the biologically active agent can be labeled such that it can be detected and / or provided in a “pro” form that is converted or modified to an active form after delivery.

  In some embodiments, the biologically active agent is a small molecule and / or an organic compound having drug activity. In some embodiments, the biologically active agent is a clinically used drug. In some embodiments, the drug is an antibiotic, antiviral agent, anesthetic agent, anticoagulant agent, anticancer agent, enzyme inhibitor, steroid agent, anti-inflammatory agent, antitumor agent, antigen, vaccine, antibody, Decongestant, antihypertensive, sedative, pregnancy regulator, progesterone, anticholinergic, analgesic, antidepressant, antipsychotic, β-anti-adrenergic, diuretic, cardiovascular, vascular Agonists, non-steroidal anti-inflammatory agents, etc. Of particular interest are biologically active substances suitable for transdermal administration.

  The biologically active substance to be delivered may be a mixture of pharmaceutically active agents. For example, a local anesthetic can be delivered with an anti-inflammatory agent such as a steroid. Local anesthetics can be administered in combination with a vasoactive agent such as epinephrine. As another example, antibiotics can be combined with inhibitors of enzymes normally made using bacteria to inactivate antibiotics (eg, penicillin and clavulanic acid).

  In some embodiments, the biologically active agent is a diagnostic agent. In some embodiments, diagnostic agents include gases; positron emission tomography (PET), computed tomography (CAT), single photon emission computed tomography, x-ray, fluoroscopy and Commercially available imaging agents used in magnetic resonance imaging (MRI); as well as contrast agents are included. Examples of suitable materials used as MRI contrast agents include gadolinium chelates and iron, magnesium, manganese, copper and chromium. Examples of materials used in CAT and X-ray imaging include iodine-based materials.

  In some embodiments, the biologically active agent is a prophylactic agent. In some embodiments, the prophylactic agent comprises a vaccine. Vaccines can include isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically engineered organisms or viruses and cell extracts. The prophylactic agent can be used in combination with an adjuvant such as interleukin, interferon, cytokine and cholera toxin, alum, Freund's adjuvant and the like. Prophylactic drugs include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyterogenes, Streptococcus pyrogenes, Streptococcus pylogenes Bacillus anthracis, Clostridium tetani, Clostridium botulism, Clostridium perfringens, Neisseria meningitidis idis), Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Bacillus pneumoniae, Hemophilus flu (Francisella tularensis), Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium tuberculum Mycobacterium leprae, Treponema pallidum, Leptospiros terrogans, Borrelia burgdorferi, Bacteria genus B And influenza B, respiratory syncytial virus, parainfluenza, measles, varicella-zoster herpes, herpes simplex type 1 and 2, cytomegalovirus, Epstein Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus , Poliovirus, mumps, rabies, Antigens of viruses such as rubella, Coxsackie virus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D and E viruses; Cryptococcus neoformans, histoplasma Capsulasum (Candida albicans), Candida tropicalis, Nocardia asteroides, Rickettica ricketik (Myco plasma pneumonia), Umm disease Chlamydia (Chlamydial psittaci), Chlamydia trachomatis (Chlamydial trachomatis), falciparum malaria parasite (Plasmodium falciparum), Trypanosoma Say (Trypanosoma brucei), Entamoeba histolytica (Entamoeba histolytica), Toxoplasma gondii (Toxoplasma gon. dii), fungal, protozoan and parasite antigens such as Trichomonas vaginalis, Schistosoma mansoni. These antigens may be in the form of complete killed bacteria, peptides, proteins, glycoproteins, carbohydrates or combinations thereof.

  In some embodiments, the biologically active agent can be a protein. As used herein, the terms “protein” and “peptide” can be used interchangeably. In certain embodiments, the peptide is an amino acid having a size in the range of about 5 to about 40, about 10 to about 35, about 15 to about 30, or about 20 to about 25. Peptides from a panel of peptides containing random sequences and / or sequences that have been altered to consistently yield a panel of maximally diverse peptides can be used.

  In some embodiments, the biologically active agent can be an antibody. In some embodiments, antibodies include, but are not limited to, polyclonal, monoclonal, chimeric (ie, “humanized”), single chain (recombinant) antibodies. In some embodiments, the antibody can have low effector function and / or bispecific molecules. In some embodiments, the antibody can comprise a Fab fragment and / or a fragment produced by a Fab expression library.

  In some embodiments, the biologically active agent can be a nucleic acid. In some embodiments, the oligonucleotide comprises DNA, RNA, mixed chimeras, derivatives, characteristic portions and / or variants thereof. The oligonucleotides of the invention may be single stranded and / or double stranded. For example, oligonucleotides can be modified with a base moiety, sugar moiety and / or phosphate backbone to improve molecular stability, hybridization, and the like.

  In certain embodiments, the nucleic acid comprises an antisense molecule that binds to a translation initiation site, transcription initiation site, and / or splice junction. Antisense oligonucleotides bind to the target mRNA and / or prevent translation. Alternatively or in addition, the antisense oligonucleotide can bind to the DNA of the target gene, eg, a regulatory element.

  In some embodiments, a nucleic acid comprising a ribozyme designed to catalytically cleave transcription of a target mRNA can be used to prevent target mRNA translation and / or target expression (eg, PCT See patent application WO 90/11364; and Sarver et al., 1990, Science 247: 1222; both of which are incorporated herein by reference).

  Alternatively or in addition, endogenous target gene expression is reduced by targeting a deoxyribonucleotide sequence complementary to the target gene's regulatory region (ie, the target gene's promoter and / or enhancer). To form a triple helix structure that blocks transcription of the target gene in target muscle cells in the body (reviewed in Helene, 1991, Anticancer Drug Des. 6: 569; Helene et al., 1992, Ann, NY Acad. Sci. 660: 27; and Maher, 1992, Bioassays 14: 807, all of which are incorporated herein by reference).

  In some embodiments, the biologically active substance is a nutritional supplement. In some embodiments, the nutritional supplement provides a basic nutritional value. In some embodiments, the nutritional supplement provides a health benefit or medical benefit. In some embodiments, the nutritional supplement is a dietary supplement.

  In some embodiments, the nutritional supplement is a vitamin. In some embodiments, the vitamin is vitamin A (retinoid), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (piroxidone), vitamin B7 ( One or more of biotin), vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D, vitamin E or vitamin K.

  In some embodiments, the nutritional supplement is a mineral. In some embodiments, the mineral is bismuth, boron, calcium, chlorine, chromium, cobalt, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, rubidium, selenium, silicon, sodium, One or more of strontium, sulfur, tellurium, titanium, tungsten, vanadium or zinc.

  In some embodiments, the nutritional supplement is an essential amino acid. In some embodiments, the amino acid is one or more of arginine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan or valine.

  In some embodiments, the nutritional supplement includes fatty acids and / or omega-3 fatty acids (eg, DHA or ARA), fruit and vegetable extracts, lutein, phosphatidylserine, lipoid acid, melatonin, glucosamine, chondroitin, aloe , Googles, green tea, lycopene, food in general, food additives, herbs, phytonutrients, antioxidants, fruit flavonoid ingredients, evening primrose oil, flaxseed oil, fish oil and marine animal oils (eg cod liver oil) and live fungi (Probiotics). In some embodiments, nutritional supplements can include biofoods (also known as “pharmaceutical foods”) that have been genetically engineered to have desired properties.

  Examples of nutritional supplements and dietary supplements are described, for example, in Roberts et al. It is disclosed. Nutritional supplements and dietary supplements can be found in Physicians 'Desk Reference for Nutritional Supplements, 1st Edition, 2001 and Physicians' Desk Reference for Herbal Medicines, 1st Edition, 2001. Is also disclosed.

  In some embodiments, inventive nanoparticles loaded with nutritional supplements can be incorporated into food products. For example, nanoparticles carrying a nutritional supplement can be dissolved in a liquid such as a beverage.

  In some embodiments, the biologically active substance is a cosmetic and / or dermatological agent. In some embodiments, the cosmetic and / or dermatological agent is a vitamin and its derivatives (eg, vitamin E and its esters, vitamin C and its esters, vitamin B, vitamin A alcohol or retinol and its esters), provitamins (Eg, panthenol, niacinamide or ergocalciferol), antioxidants, phenolic compounds (eg, benzoyl peroxide), essential oils, wetting agents, sunscreens, humectants, proteins, ceramides and pseudoceramides it can.

In some embodiments, the biologically active agent may be one or more botulinum toxin peptides or protein complexes. In some embodiments, the botulinum toxin may be one or more of botulinum toxin serotypes A, B, C ₁ , C ₂ , D, E, F, or G. In some embodiments, the botulinum toxin may be an isolated and / or purified botulinum toxin. In some embodiments, the botulinum toxin may be a partially isolated and / or partially purified botulinum toxin. In some embodiments, the botulinum toxin may be a natural botulinum complex. In some embodiments, the botulinum toxin may be associated with a non-toxic protein. In some embodiments, the botulinum toxin may be a recombinant botulinum toxin.

Those skilled in the art will appreciate that this is an exemplary list of biologically active substances, but not exhaustive. Any biologically active agent can be encapsulated in or bound to the surface of the nanoparticle.
Release-retarding agent In some embodiments of the invention, particularly in embodiments comprising one or more biologically active agents (eg, unmodified peptides), the nanoparticle compositions of the invention provide control of that agent. It further comprises or is formulated with one or more release-retarding components that allow release. Any release-retarding component known in the art is suitable for use in making the nanoparticles of the present invention. In some embodiments, the release-retarding component is a hydrophilic and / or hydrophobic polymer. Release-retarding components include, for example, cellulose or its derivatives, acrylic polymers, ester polymers, polymers based on vinyl pyrrolidone, rubbers, other natural polymers and / or combinations thereof.

  In some embodiments, the release-retarding component is cellulose or a derivative thereof. In certain embodiments, the cellulose or derivative thereof comprises one or more of hydroxypropyl methylcellulose, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. In certain embodiments, the cellulose or derivative thereof is methylcellulose or a derivative thereof. In certain embodiments, the cellulose or derivative thereof is hydroxypropyl methylcellulose (HPMC). One skilled in the art will appreciate that other cellulose polymers can be used, including other alkyl cellulose polymers.

  In some embodiments, the release-retarding component is an acrylic polymer. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), methacrylic acid. Acid alkylamide copolymer, poly (methyl methacrylate), poly (methacrylic anhydride), methyl methacrylate, polymethacrylate, poly (methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymer and one or more of the above polymers Combinations containing are included. The acrylic polymer can comprise a fully polymerized copolymer of acrylic acid and methacrylic acid ester having a low content of quaternary ammonium groups.

  In some embodiments, the release-retarding component is a polyester. In some embodiments, the polyester includes polyalkylene glycol, poly (glycolide-co-lactide), PEGylated poly (lactic acid-co-glycolic acid), poly (lactic acid), PEGylated poly (lactic acid), poly ( Glycolic acid), PEGylated poly (glycolic acid), copolymers of polylactic acid and polyglycolic acid, and derivatives thereof. In some embodiments, the polyester includes, for example, polyanhydride, poly (orthoester) PEGylated poly (orthoester), poly (caprolactone), PEGylated poly (caprolactone), polylysine, PEGylated polylysine, poly (ethylene). Imine), PEGylated poly (ethyleneimine) and derivatives thereof. In some embodiments, the polyester includes, for example, polycaprolactone, poly (L-lactide-co-L-lysine), poly (serine ester), poly (4-hydroxy-L-proline ester), poly [α -(4-aminobutyl) -L-glycolic acid] and its derivatives.

  In some embodiments, the release-retarding component is a crosslinked polymer of poly (vinyl-pyrrolidone). In some embodiments, the polymer is crospovidone. In some embodiments, the polymer is non-crosslinked poly (vinyl-pyrrolidone). In some embodiments, the polymer is povidone.

  In some embodiments, the release-retarding component may be a natural polymer. In some embodiments, the natural polymer is a gum that includes, for example, xanthan gum, alginic acid, karaya gum, sodium alginate and / or locust bean gum. In some embodiments, the natural polymer can be a protein (eg, albumin), lipid, nucleic acid, or carbohydrate and the release-retarding component is a polyester. In some embodiments, the polyester includes polyalkylene glycol, poly (glycolide-co-lactide), PEGylated poly (lactic acid-co-glycolic acid), poly (lactic acid), PEGylated poly (lactic acid), poly ( Glycolic acid), PEGylated poly (glycolic acid), copolymers of polylactic acid and polyglycolic acid, and derivatives thereof. In some embodiments, the polyester includes, for example, polyanhydride, poly (orthoester) PEGylated poly (orthoester), poly (caprolactone), PEGylated poly (caprolactone), polylysine, PEGylated polylysine, poly (ethylene). Imine), PEGylated poly (ethyleneimine) and derivatives thereof. In some embodiments, the polyester includes, for example, polycaprolactone, poly (L-lactide-co-L-lysine), poly (serine ester), poly (4-hydroxy-L-proline ester), poly [α -(4-aminobutyl) -L-glycolic acid] and its derivatives.

In some embodiments, the release-retarding component is a crosslinked polymer of poly (vinyl-pyrrolidone). In some embodiments, the polymer is crospovidone. In some embodiments, the polymer is non-crosslinked poly (vinyl pyrrolidone). In some embodiments, the polymer is povidone. In some embodiments, the release-retarding component may be a natural polymer. In some embodiments, the natural polymer is a gum that includes, for example, xanthan gum, alginic acid, karaya gum, sodium alginate, and / or locust bean gum. In some embodiments, the natural polymer may be a protein (eg, albumin), lipid, nucleic acid, or carbohydrate.
Formulations The nanoparticle compositions of the present invention can be formulated for administration to a subject. In certain embodiments, the nanoparticle composition of the present invention is formulated to be applied to the skin to achieve transdermal delivery to a subject. For example, the nanoparticle compositions of the present invention can be formulated into cosmetic or other formulations that are applied topically.

  Human skin includes the dermis and epidermis. The epidermis has multiple layers of tissue: the stratum corneum, the transparent layer, the granule layer, the spiny layer and the basal layer (which are in turn from the outer surface of the skin to the inside). The stratum corneum usually provides the most important obstacle in transdermal delivery of pharmaceuticals (possibly especially in the case of unmodified peptides). The stratum corneum is usually about 10-15 μm thick and consists of keratinocytes (keratinocytes) arranged in flat layers. The intercellular space between corneocytes is filled with lipidic structures and can play an important role in the penetration of substances through the skin (Bauerova et al., 2001, European Journal of Drugs and Pharmacokinetics, 26:85; which is incorporated herein by reference).

  The rest of the epidermis under the stratum corneum is about 150 μm thick. The dermis is about 1 mm to 2 mm thick and is located below the epidermis. The dermis is innervated by various capillaries and neurites.

  Previous attempts to administer pharmaceuticals transdermally have focused on increasing the permeability of the stratum corneum. Some attempts have included the use of chemical accelerators that increase the permeability of molecules through the skin. Some attempts have included using mechanical devices to bypass or remove a portion of the stratum corneum. In addition, some attempts have included using ultrasound or iontophoresis to facilitate the penetration of the drug through the skin. In most cases, what is aimed at is generally a drug that passes through the skin, typically a small molecule, where the drug is delivered to the capillary bed in the dermis where the drug can be systemically taken into the subject to achieve a therapeutic effect. there were.

  The present invention provides, inter alia, a method for transdermally administering an unmodified peptide that does not require the use of an abrading agent or disintegrant (either chemical, mechanical, electrical, magnetic, etc.). To do. Surprisingly, we have found that the botulinum toxin incorporated in the nanoparticle composition of the present invention does not require further steps to make the stratum corneum permeable or disrupt it, It has been found that it can be effectively delivered transdermally. The use of such agents or steps with the botulinum nanoparticle compositions of the present invention is not necessarily excluded in all embodiments of the present invention, nor is it necessarily required.

  Accordingly, the present invention provides a method of administering an unmodified peptide by topical application of the nanoparticle composition of the present invention. In some embodiments, the nanoparticle composition of the present invention is applied directly to the skin for absorption through the epidermal layer. In some embodiments, the nanoparticle composition removes the stratum corneum, skin pores and / or skin glands without the use of chemical or mechanical skin penetration enhancers or other agents that cause abrasion. It can penetrate the top layer of the skin it contains.

  Those skilled in the art will recognize that the compositions of the present invention for topical administration are emollients, nutritional lotion type emulsions, cleansing lotions, cleansing creams, emulsions, emollient lotions, massage creams, emollient creams, makeup bases, lipsticks, As a cosmetic formulation such as facial packs or facial gels, shampoos, rinses, body cleansers, detergent formulations such as hair art or soap, or dermatological compositions such as lotions, ointments, gels, creams, patches or sprays It will be understood that it can be prepared.

  Such formulations of the nanoparticle compositions of the present invention are generally in combination with one or more excipients such as, for example, fillers, sequestering agents, softeners, coloring materials (eg, pigments and dyes) and perfumes. including.

  In some embodiments, the nanoparticle composition of the present invention is formulated as a cream. The term “cream” refers to a spreadable composition that is usually formulated for application to the skin. Creams generally comprise an oil and / or fatty acid based matrix. Creams formulated according to the present invention can include nanoparticles and can allow substantially complete penetration (eg, of such nanoparticles) through the skin when administered topically. Such creams can also act as carriers for incorporated materials (eg, for botulinum toxins).

  One skilled in the art will appreciate that the nanoparticle compositions of the present invention can be incorporated into devices such as patches, for example.

  A variety of transdermal patch structures are known to those skilled in the art, and those skilled in the art will appreciate that the nanoparticle compositions of the present invention can be readily incorporated into any of such various structures. In some embodiments, the transdermal patch can further include a plurality of needles extending from one side of the patch applied to the skin. The needle extends from the patch and protrudes through the stratum corneum of the skin. In some embodiments, the needle does not break a blood vessel.

  In some embodiments of the invention, the nanoparticle composition is placed in a depot in the patch so that the pressure applied to the patch causes the unmodified peptide to be directed out of the patch (optionally through a needle), Can pass through the stratum corneum.

  In some embodiments of the invention, the transdermal patch includes an adhesive. Some examples of adhesive patches are well known (eg, US Pat. Nos. 296,006, 6,010,715, 5,591,767, 5, 008,110, 5,683,712, 5,948,433, and 5,965,154, all of which are incorporated herein by reference). Adhesive patches are generally characterized by having an adhesive layer stretched over a person's skin, a depot or reservoir for holding the drug, and an external surface that prevents the drug from leaking from the depot. The outer surface of the patch is non-adhesive.

  One skilled in the art will appreciate that a transdermal patch is just one example of a device with which the nanoparticle composition of the present invention can be administered. To give some other examples, there is a device that allows the composition to be applied without first applying the composition to a person's finger (which can lead to undesirable finger paralysis). Can be used. Suitable devices include spatulas, swabs, needleless syringes and adhesive patches. The use of a spatula or swab or the like necessitates insertion of such a device into a container containing the composition. The syringe can be used by filling the syringe with the composition. The composition can then be spread topically with a spatula or swab or extruded from a syringe onto a person's skin.

In many embodiments of the invention, it is desirable to limit the delivery of the unmodified peptide to only the delivery region of interest. In some embodiments, such limited delivery is achieved by applying the nanometers of the present invention with an applicator device that allows the composition to be applied to a target site of skin without applying the composition to a non-target site region of skin. It can be realized by using a particle composition. Obviously, transdermal patches can be used for this purpose. Alternatively or in addition, if the modified peptide is to be applied topically only to selected areas, other areas can be covered or pretreated or protected from exposure.
Examples The following examples only provide examples of specific embodiments contemplated by the present invention. These examples are not intended to be limiting in any way.

Example 1
Pentapeptide Nanoparticle Formulation This example comprises a nanoemulsion comprising the pentapeptide, KTTKS (SEQ ID NO: 1) (Katayama et al., Supra), known to have biological activity against skin structure. Peptide nanoparticle compositions are provided.

A pentapeptide nanoemulsion formulation was prepared as follows.
• 800 mg soybean oil and 800 mg Tween 80 were stirred in a sterile vial for 5 minutes.
-8.4 ml of water containing 0.0001 g of peptide KTTTS (SEQ ID NO: 1) was added and stirred for 20 minutes.
• The sample was homogenized for 1 minute.
• The sample was stirred for 20 minutes.
• Sample was microfluidized once at 23,000 psi.

  The resulting pentapeptide nanoemulsion was evaluated for particle size using a Malvern Nano S type particle sizer that can measure particles of about 0.6 nm to 6000 nm. The pentapeptide nanoemulsion formulation had two particle size peaks and an average particle size of 106 nm (Table 2).

。

.

(Example 2)
Pentapeptide Nanoparticle Formulation with Biological Effect and Transcutaneous Osmosis This example illustrates the pentapeptide KTTKS (SEQ ID NO: 1), which is known to have biological activity against the structure of the skin. Provided are peptide nanoparticles comprising a nanoemulsion comprising Katayama et al., Supra; which is incorporated herein by reference. This example demonstrates the biological efficacy on the skin of transdermally applying peptide nanoparticles, in this case KTTKS (SEQ ID NO: 1).
Materials and Methods Pentapeptide nanoemulsion formulations were prepared as follows.
5.6 g of Labrafac WL1349 oil and 5.6 g of Tween 80 were stirred for 5 minutes in a sterile beaker.
In a separate beaker, 58.8 g of reagent grade water was placed. 0.010 g of peptide KTTTS (SEQ ID NO: 1) was added to the water and stirred for 20 minutes.
Add the contents of the first beaker to the contents of the beaker (ie the water and peptide) and then stir for 20 minutes.
• The entire sample was microfluidized once at 23,000 psi.

  The particle diameter of the obtained pentapeptide nanoemulsion was evaluated using a Malvern Nano S type particle size measuring device capable of measuring particles of about 0.6 nm to 6000 nm. The pentapeptide nanoemulsion formulation had an average particle size of 114.4 nm. About 95% of the particles were less than 130 nm in size.

  Next, the pentapeptide nanoemulsion was mixed with an equal volume of skin cream (base PCCA burnishing cream light) and stirred with Vorlux to make a uniform cream to obtain a “treatment cream”.

  A “control cream” was prepared in the same manner as the therapeutic cream except that no peptide was added during the process.

  Ten Swiss Webster mice, each weighing about 20 g, were purchased. Once in stock, all of these mice were acclimated in the cage for 1 week (5 mice per group were grouped as defined below) and standard bedbed (cage bedding) ) And Purina 5001 chow meal. One week later, the following treatment paradigm was applied.

Treatment Paradigm Group 1 (Control): Every 8 weeks, 75 μl of control cream was applied to each of 5 mice on its back with gloved fingers until no cream was observed. Two days before the first treatment, the mouse's back was shaved with an electric razor at weekly intervals.

  Group 2 (Treatment): Every 8 weeks, 75 μl of treatment cream was applied to the back of each mouse with gloved fingers until no cream was observed. Two days before the first treatment, the mouse's back was shaved with an electric razor at weekly intervals.

Evaluation The skin from the back of each mouse treated with either a control cream or a therapeutic cream was preserved and then treated with Masson's trichrome tissue stain. Staining intensity was evaluated at 400 × magnification using a histological scale of 1 to 4 for staining intensity. 1 = Unstained and collagen fibrils are very thin. 2 = Minimum collagen fibril width with minimal staining. 3 = Medium fibril width with moderate staining. 4 = Wide fibril with strong staining.

Results Histological Evaluation The average histological score of skin tissue stained with Masson's trichrome staining was 2.33 out of 4 possible in the control group. By comparison, the average histological score of stained skin of the treatment group mice was 3.67 out of 4 possible. This shows that the collagen-staining intensity of the treatment group is increased by 57% over the control group. See, for example, micrographs of skin tissue specimens from each of the control and treatment groups in FIG.

Evaluation of skin thickness effect Each skin was measured by measuring the skin thickness by skin layer thickness inspection method according to the histological cross-section test of the skin of the back of the mouse, which was sliced and placed on a slide glass. Determine the depth (in mm) of the layer.

Assessment of extracellular matrix production Collagen is a major component of the contents of the extracellular matrix. Collagen content was sliced and placed on slide galley using two separate tissue staining methods for collagen (Picrosirius Red and Pterocarpus Osun) Assess in two separate tests of the skin on the back of mice.

  In order to detect the hydroxyproline content of the homogenized formulation of the mouse back skin, the collagen content is assessed using Western blotting. Hydroxyproline content is representative of collagen content.

Conclusion The results show that, on average, treatment groups have statistically more collagen than the control group by histological evaluation. These controlled data indicate that topical pentapeptide nanoemulsion formulations have measurable biological effects on the skin compared to control creams that do not contain such pentapeptides. ing. Previous studies have shown that peptides cannot penetrate intact skin without chemical modification (Katayama et al., Supra). Thus, these data indicate that the nanoemulsion formulation of the present invention allows the penetration of unmodified peptides, increases collagen production in the skin and, consequently, increases the skin thickness, the known biology of peptides. It is shown to bring about an effect on the skin that is consistent with the physical effects.

  The results appear to on average indicate that the treatment group has statistically thicker skin than the control group. The results appear to show that, on average, the treatment group has statistically more collagen than the control group, as measured by two tissue staining methods and Western probing with hydroxyproline.

(Example 3)
Effect of transcutaneous application of peptide nanoparticles as a skin thickening and extracellular matrix stimulant on mice: effect of changing the concentration of peptide in nanoparticles This example This demonstrates the effect of varying the peptide concentration on the biological efficacy of skin with peptide nanoparticles applied transdermally.

Materials and Methods The experiment described in Example 3 is repeated except that the concentration of peptide in the therapeutic cream is increased by an order of magnitude or reduced by an order of magnitude.

Results and Conclusions The results appear to show that on average, mice treated with an order of magnitude higher peptide concentration have statistically thicker skin than mice treated with higher peptide concentrations. Results show that, on average, mice treated with an order of magnitude higher peptide concentration have a statistically higher amount of collagen than mice with lower peptide concentrations, as measured by two tissue staining methods and Western blot of hydroxyproline. It seems to indicate that That is, these controlled data may suggest that the biological effects of peptide nanoemulsions on the skin depend on the concentration of peptide incorporated.

Example 4
Administration of Pentapeptide Nanoparticles to a Human Subject to Reduce Skin Lines This example demonstrates the biological efficacy of human skin that has been coated with peptide nanoparticles transdermally.

Materials and Methods The pentapeptide nanoemulsion prepared in Example 1 or 2 is prepared and mixed with an equal volume of skin cream (base PCCA burnishing cream light) and then vortexed into a uniform cream to produce “treatment cream "Agent".

  The “non-nanotherapy cream” was mixed with the same amount of pentapeptide as in Example 1 in the same amount of water, then vortexed with the same amount of skin cream used to prepare the therapeutic cream. Prepare.

  A “control cream” is prepared by vortexing the same amount of water as in Example 1 or 2 and the same amount of skin cream used to prepare the therapeutic cream.

  Thirty healthy human subjects with prominent facial wrinkle lines (such as those seen in people with light-damaged skin) are controlled with a double-blind placebo using left-right randomization To the face division test. All subjects are rated on a 5-point scale by an observer who has made the treatment status invisible. A scale score of 0 is normal skin and a score of 5 is a severe facial wrinkle line and wrinkles (mainly around the eyes or in the “eye corners” area). Cheek skin texture is also evaluated with respect to pore size (from small to large) and smoothness (from smooth to rough / crushed). Register the subject only if they have a score of 2.5 or higher in the initial examination. Take a picture of the subject's face with standardized field of view, distance and lighting conditions.

Treatment Paradigm The patient agrees not to use any facial skin care products for 3 weeks other than a control cream that can be used twice a day at 12 hour intervals. After this initial “wash-out” period, each patient is given two cream tubes labeled “Right” and “Left” with a unique numeric code for each tube. They are instructed to use the right tube on the right side of the face and the left tube on the left side of the face twice a day at 12 hour intervals. Instruct them to apply a “bean size” amount of cream (about 0.4 g) on both sides of the face. They are also instructed not to use other facial skin care products. For 10 of the subjects (control group), the right tube contains the control cream and the left tube contains the control cream. For 10 of the subjects (non-nanotherapy group), the right tube contains the control cream and the left tube contains the non-nanotherapy cream. For 10 of the subjects (nanotherapy group), the right tube contains the control cream and the left tube contains the nanotherapy cream.

Evaluation Following the wash-out period, subjects are observed and photographed at 4, 8 and 12 weeks after starting the treatment protocol. Furthermore, the observer who made the treatment state of the object invisible, and the object itself score the skin texture on the above scale for each of the right and left sides of the face.

Results and Conclusions Results show that, on average, the nanotreatment group is statistically greater than the difference observed between the right and left scores for the control and non-nanotherapy groups, the right and left facial texture scores It seems to indicate that there is a difference between them (indicating improved skin appearance). That is, these controlled data show that a topical pentapeptide nanoemulsion formulation has a simple cream with a control cream that does not have such a pentapeptide, and the same pentapeptide that was not present in the nanoparticle formulation. It appears to have a measurable cosmetic effect on the skin when compared to a cream (non-nano cream).

Equivalents and Scope The foregoing has described certain non-limiting preferred embodiments of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Those skilled in the art will recognize that various changes and modifications can be made to the description of the invention without departing from the spirit or scope of the invention as defined in the following claims.

  In the claims, articles such as “a”, “an” and “the” mean one or more than one unless otherwise specified or apparent from the context. . A claim or detailed description that includes “or” between one or more members of a group, unless specified otherwise, or apparent from context, does not One, more than one, or all of these shall be satisfied if they are present in, used in, or related to a given product or process. The invention includes embodiments in which exactly one member of the group is present in, employed in, or associated with a given product or method. The invention also includes embodiments in which more than one or all of the group members are present in, used in, or associated with a given product or method. Furthermore, one or more limitations, elements, sections, descriptive terms, etc., from one or more of the claims or from the relevant part of the description are introduced into another claim. It should be understood that all modifications, combinations and substitutions are encompassed. For example, any claim that is dependent on another claim can be modified to include one or more of the limitations recited in any other claim that is dependent on the same basic claim . Further, if a composition is described in the claims, the present description unless otherwise specified or unless it is clear to a person skilled in the art that a conflict or inconsistency will occur. A method of using the composition for any of the purposes disclosed herein, and the composition by any of the methods of making disclosed herein, or other methods known in the art It should be understood that these methods are included. For example, any of the compositions of the present invention may form, progress in adhesion due to any of a plurality of locations and / or due to any of the causes discussed herein or known in the art. It should be understood that and / or can be used to prevent recurrence. Any of the compositions made by the method of preparing a composition disclosed herein may be of any of a plurality of locations and / or as discussed herein or known in the art. It should also be understood that it can be used to prevent the formation, progression and / or recurrence of adhesions due to either. Furthermore, the present invention encompasses compositions made by any of the methods for preparing compositions disclosed herein.

  When an element is shown as an enumeration, for example in the Markush group format, it should be understood that each subgroup of that element is also disclosed and any element can be excluded from that group. It should also be noted that the term “comprising” is intended to be non-limiting and allows the inclusion of additional elements or steps. In general, where the invention, or aspects of the invention, are referred to as including particular elements, features, steps, etc., the particular embodiments of the invention or aspects of the invention are inferred from such elements, features, steps, etc. It should be understood that it consists of or consists essentially of. For the sake of brevity, such embodiments are not specifically illustrated herein in this language. Thus, for each embodiment of the invention that includes one or more elements, features, steps, etc., the invention also provides embodiments that consist of, or consist essentially of, such elements, features, steps, etc.

  Where ranges are indicated, this includes endpoints. Further, unless otherwise specified or apparent from the context and / or from the understanding of one of ordinary skill in the art, values given as ranges are not intended to indicate different embodiments of the invention, unless expressly specified otherwise by the context. It should be understood that any specific value up to one tenth of the lower limit unit of the range can be envisaged within the indicated range. Unless otherwise specified or apparent from the context and / or from the understanding of one of ordinary skill in the art, a value given as a range can assume any subrange within the given range, and the endpoint of that subrange is It should also be understood that it is expressed with the same accuracy as one tenth of the lower limit unit of the range.

  Furthermore, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application or aspect of the compositions and / or methods of the invention (eg, any peptide, any peptide modification, any nanoparticle, any nanoemulsion, any surfactant, Any oil, any premix component, any nanoemulsion preparation method, any treatment method, etc.) can be excluded from any one or more claims. For the sake of brevity, not all embodiments in which one or more elements, features, objects or aspects are excluded are expressly set forth herein.

Claims (52)

A suspension or dispersion of nanoparticles comprising a population of particles, have a diameter of predominantly 1 0 to 3 nm of the particles, wherein the nanoparticles, biological skin, subcutaneous tissue or adjacent muscle Suspension or dispersion comprising at least one unmodified peptide of 5 to 30 amino acids in length comprising the amino acid sequence KTTKS having a specific activity. Mostly, suspension or dispersion according to claim 1 having a diameter in the range of 1 0 to 2 50 nm of the particles. Mostly, suspension or dispersion according to claim 1 having a diameter in the range of 1 0 to 2 nm of the particles. Mostly, suspension or dispersion according to claim 1 having a diameter of 1 0 to 1 50 nm range of the particles. Mostly, suspension or dispersion according to claim 1 having a diameter of 1 0 to 1 20 nm range of the particles. Mostly, suspension or dispersion according to claim 1 having a diameter in the range of 1 0 to 1 nm of the particles. Mostly, suspension or dispersion according to claim 1 having a diameter in the range of 1 0 to 5 0 nm of the particles.   The suspension or dispersion according to claim 1, wherein the particles have an average particle size in the range of 10 to 250 nm.   The suspension or dispersion according to claim 1, wherein the particles have an average particle size in the range of 10 to 200 nm.   The suspension or dispersion according to claim 1, wherein the particles have an average particle size in the range of 10 to 150 nm.   The suspension or dispersion according to claim 1, wherein the particles have an average particle size in the range of 10 to 120 nm.   The suspension or dispersion according to claim 1, wherein the particles have an average particle size in the range of 10 to 100 nm.   The suspension or dispersion according to claim 1, wherein the particles have an average particle size in the range of 10-50 nm.   The suspension or dispersion according to claim 1, wherein the composition comprises 0% to 30% of an oil component.   The suspension of claim 1, wherein the nanoparticles comprise an amino acid sequence selected from the group comprising EYKTTKSSRL, GKTVIEYKTTTS, GKTVIEYKTTTSSRL, WGKTVIEYKTTTSSRLPIID, CTSHTGAWGKTVIEYKTTTS, or combinations thereof. Before SL nanoparticles comprise amino acid sequence E YKTTKSSRL, suspension or dispersion according to claim 1. The suspension or dispersion according to any one of claims 1 to 16 , wherein the suspension or dispersion contains hyaluronic acid. Before SL nanoparticles comprise amino acid sequence G KTVIEYKTTKS, suspension or dispersion according to claim 1. Before SL nanoparticles comprise amino acid sequence G KTVIEYKTTKSSRL, suspension or dispersion according to claim 1. Before SL nanoparticles comprise amino acid sequence W GKTVIEYKTTKSSRLPIID, suspension or dispersion according to claim 1. Before SL nanoparticles comprise amino acid sequence C TSHTGAWGKTVIEYKTTKS, suspension or dispersion according to claim 1. The suspension or dispersion according to any one of claims 1 to 21 , wherein the nanoparticles are nanoemulsions. It said nanoemulsion comprises at least one oil, at least one surfactant, suspension or dispersion according to claim 2 2, including water and non-modified peptide. The nanoemulsions, single oil, a single surfactant, suspension or dispersion according to claim 2 2, including water and non-modified peptide. The oil is almond, apricot, avocado, babas, bergamot, black currant seed, borage, cadet, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed , Emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, loofah, grape seeds, hazelnuts, hyssop, jojoba, kukui seeds, lavandin, lavender, lemon, lychee aqaba, macadamia nuts, aoi, mango seeds, meadowweed seeds , Mink, nutmeg, olive, orange, orange luffy, palm, palm kernel, peach seed, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasanqua, savory, sea buckthorn, Oil of maize, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut and wheat germ; butyl stearate; caprylic acid triglyceride; capric acid triglyceride; cyclomethicone; diethyl sebacate; dimethicone 360; 3. An isopropyl myristate; a mineral oil; an octyldodecanol; an oleyl alcohol; a silicone oil; a short chain triglyceride; a medium chain triglyceride; a Labrafac WL1349; a long chain triglyceride; its saturated oil; and its unsaturated oil. suspension or dispersion according to the three 21 to 24 any one of. Suspension or dispersion according to any one of claims 2 3 21 to 24 wherein the oil is soybean oil. Suspension or dispersion according to any one of claims 2 3 21 to 24 wherein the oil is a medium chain triglyceride. Suspension or dispersion according to any one of claims 2 3 21 to 24 wherein the oil is a Labrafac WL1349. The surfactant is phosphoglyceride; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; Diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surface active fatty acids such as palmitic acid or oleic acid; fatty acids; fatty acids Monoglycerides; fatty acid dinoglycerides; fatty acid amides; sorbitan trioleate (Span 85) Polysorbate; sorbitan monolaurate (Span20); polysorbate 20 (Tween-20); polysorbate 60 (Tween-60); polysorbate 65 (Tween-65); polysorbate 80 (Tween-80); polysorbate 85 (Tween-85); Surfactin; poloxamer; sorbitan fatty acid esters such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; Dipalmitoyl phosphatidylglycerol; stearylamine; dodecylamine; Oxadecyl-amine; Acetyl palmitate; Glycerol ricinoleate; Hexadecyl stearate; Isopropyl myristate; Tyloxapol; Poly (ethylene glycol) 5000-phosphatidylethanolamine; Poly (ethylene glycol) 400-monostearate; Phospholipids; High synthetic and / or natural detergents having surfactant properties; deoxycholate; suspension of and according to any one of claims 2 3 21 to 24 which is selected from the group consisting of ion pairing agents; cyclodextrin; chaotropic salt Suspension or dispersion. Suspension or dispersion according to any one of claims 2 3 21 to 24 wherein the surfactant is a Tween. Surfactant and the weight ratio of oil 0.5: 21 to: a suspension or dispersion according to any one of claims 2 3-3 0 1 by weight. The weight ratio of surfactant to oil is 0 . 25: The suspension or dispersion according to any one of claims 2 3-3 0 1. The weight ratio of surfactant to oil is 0: 5: suspension or dispersion according to any one of claims 2 3-3 0 1. The weight ratio of surfactant to oil is 1: The suspension or dispersion according to any one of claims 2 3-3 0 1. The weight ratio of surfactant to oil is 2: The suspension or dispersion according to any one of claims 2 3-3 0 1. The weight ratio of surfactant to oil is 3: The suspension or dispersion according to any one of claims 2 3-3 0 1. The suspension or dispersion according to any one of claims 1 to 21 , wherein the nanoparticles are nano micelles. 38. The method of any one of claims 1 to 37 , further comprising at least one additional ingredient that, together with the suspension or dispersion, produces a cream, oil, ointment, gel, spray, lipstick or sunscreen. Suspension or dispersion of A composition for providing utility in cosmetic conditions or treatment conditions of the skin, the composition comprising a suspension or dispersion according to any one of claims 1 to 38 effective amount. A composition for thickening the skin or subcutaneous tissue, comprising an effective amount of the suspension or dispersion according to any one of claims 1 to 38 . A composition for treating, reducing, improving, alleviating fine skin lines, delaying its development, preventing its progression, reducing its severity, or reducing its incidence there, the compositions comprising a suspension or dispersion according to any one of claims 1 to 38 effective amount. A composition for treating, reducing, improving, alleviating facial wrinkles, delaying its development, preventing its progression, reducing its severity, or reducing its incidence a composition comprising a suspension or dispersion according to any one of claims 1 to 38 effective amount. A composition for improving the appearance of a subject's skin, comprising an effective amount of the suspension or dispersion according to any one of claims 1 to 38 . A composition for maintaining the appearance of the skin of a subject, comprising an effective amount of the suspension or dispersion according to any one of claims 1 to 38 . The composition is administered using a device that allows the suspension or dispersion to be applied to a target site on the skin without applying the suspension or dispersion to a non-target site on the skin. 45. Composition according to any one of claims 39 to 44 , characterized in that 46. Composition according to any one of claims 39 to 45 , characterized in that all of the unmodified peptide penetrates the skin. 46. Composition according to any one of claims 39 to 45 , characterized in that at least 95% of the unmodified peptide penetrates the skin. 46. Composition according to any one of claims 39 to 45 , characterized in that at least 75% of the unmodified peptide penetrates the skin. 46. Composition according to any one of claims 39 to 45 , characterized in that at least 50% of the unmodified peptide penetrates the skin. 46. Composition according to any one of claims 39 to 45 , characterized in that at least 25% of the unmodified peptide penetrates the skin. 46. Composition according to any one of claims 39 to 45 , characterized in that at least 10% of the unmodified peptide penetrates the skin. 46. Composition according to any one of claims 39 to 45 , characterized in that at least 1% of the unmodified peptide penetrates the skin.

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